Method for preparing antibodies having  improved properties

ABSTRACT

The present invention is directed to methods and compositions for the production of Fc-containing polypeptides having improved properties.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions for theproduction of Fc-containing polypeptides which are useful as human oranimal therapeutic agents.

BACKGROUND OF THE INVENTION

Therapeutic proteins often achieve their therapeutic benefit throughengagement, or binding, to an endogenous protein or physiologicalcomponent to effect a desired response. For example, monoclonalantibodies often achieve their therapeutic benefit through two bindingevents. First, the variable domain of the antibody binds a specificprotein on a target cell, for example CD20 on the surface of cancercells. This is followed by recruitment of effector cells such as naturalkiller (NK) cells that bind to the constant region (Fc) of the antibodyand destroy cells to which the antibody is bound. This process, known asantibody-dependent cell cytotoxicity (ADCC), depends on a specificN-glycosylation event at Asn 297 in the Fc domain of the heavy chain ofIgG1s, Rothman et al., Mol. Immunol. 26: 1113-1123 (1989). Antibodiesthat lack this N-glycosylation structure still bind antigen but cannotmediate ADCC, apparently as a result of reduced affinity of the Fcdomain of the antibody for the Fc Receptor FcγRIIIa on the surface of NKcells.

The presence of N-glycosylation not only plays a role in the effectorfunction of an antibody, the particular composition of the N-linkedoligosaccharide is also important for its end function. The lack offucose or the presence of bisecting N-acetyl glucosamine has beenpositively correlated with the potency of the ADCC, Rothman (1989),Umana et al., Nat. Biotech. 17: 176-180 (1999), Shields et al., J. Biol.Chem. 277: 26733-26740 (2002), and Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003). There is also evidence that sialylation in the Fcregion is positively correlated with the anti-inflammatory properties ofintravenous immunoglobulin (IVIG). See, e.g., Kaneko et al., Science,313: 670-673, 2006; Nimmerjahn and Ravetch., J. Exp. Med., 204: 11-15,2007.

Given the utility of specific N-glycosylation in the function andpotency of antibodies, a method for modifying the composition ofN-linked oligosaccharides in antibodies to modify their function wouldbe desirable. In particular, it would be desirable to modify thecomposition of N-linked oligosaccharides in order to confer toFc-containing peptides, such as antibodies, an increased or enhancedability of activating immune cells. Such antibodies could be used totreat infectious diseases or neoplastic diseases as well as to serve asan adjuvant for vaccines.

Yeast and other fungal hosts are important production platforms for thegeneration of recombinant proteins. Yeasts are eukaryotes and,therefore, share common evolutionary processes with higher eukaryotes,including many of the post-translational modifications that occur in thesecretory pathway. Recent advances in glycoengineering have resulted incell lines of the yeast strain Pichia pastoris with genetically modifiedglycosylation pathways that allow them to carry out a sequence ofenzymatic reactions, which mimic the process of glycosylation in humans.See, for example, U.S. Pat. Nos. 7,029,872, 7,326,681 and 7,449,308 thatdescribe methods for producing a recombinant glycoprotein in a lowereukaryote host cell that are substantially identical to their humancounterparts. Human-like sialylated bi-antennary complex N-linkedglycans like those produced in yeast from the aforesaid methods havedemonstrated utility for the production of therapeutic glycoproteins.Thus, a method for further modifying or improving the production ofantibodies in yeasts such as Pichia pastoris would be desirable.

SUMMARY OF THE INVENTION

The invention comprises a method of enhancing an immune response in asubject in need thereof comprising: administering to the subject atherapeutically effective amount of an Fc-containing polypeptidecomprising an increased amount of α-2,3-linked sialic acid compared tothe amount of α-2,3-linked in a parent polypeptide. In one embodiment,the subject has, or is at risk of developing, an infectious disease or aneoplastic disease.

In one embodiment, the amount of α-2,3-linked sialic acid is increased(compared to the amount of α-2,3-linked in a parent polypeptide) byintroducing one or more mutations in the Fc region of the Fc-containingpolypeptide.

In one embodiment, the amount of α-2,3-linked sialic acid is increased(compared to the amount of α-2,3-linked in a parent polypeptide) byexpressing the Fc-containing polypeptide in a host cell that has α-2,3sialic acid transferase. In another embodiment, the amount ofα-2,3-linked sialic acid is increased (compared to the amount ofα-2,3-linked in a parent polypeptide) by expressing the Fc-containingpolypeptide in a host cell that has been transformed with a nucleic acidencoding an α-2,3 sialic acid transferase. In one embodiment the hostcell is a mammalian cell. In one embodiment, the host cell is a lowereukaryotic host cell. In one embodiment, the host cell is fungal hostcell. In one embodiment, the host cell is Pichia sp. In one embodiment,the host cell is Pichia pastoris.

In one embodiment, the amount of α-2,3-linked sialic acid is increased(compared to the amount of α-2,3-linked in a parent polypeptide) byintroducing one or more mutations in the Fc region of the Fc-containingpolypeptide and by expressing the Fc-containing polypeptide in a hostcell that has been transformed with a nucleic acid encoding an α-2,3sialic acid transferase.

In one embodiment, the invention comprises a method of enhancing animmune response in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of an Fc-containingpolypeptide comprising sialylated N-glycans, wherein the sialic acidresidues in the sialylated N-glycans contain α-2,3 linkages, and whereinat least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureselected from the group consisting ofSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, the sialicacid residues in the sialylated N-glycans are attached exclusively viaα-2,3 linkages. In one embodiment, the subject has, or is at risk ofdeveloping, an infectious disease or a neoplastic disease. In oneembodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, at least 30%, 40%,50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, atleast 80% of the N-glycans on the Fc-containing polypeptide comprise anN-linked oligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In any of the above embodiments, the SA couldbe NANA or NGNA, or an analog or derivative of NANA or NGNA. In oneembodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, the N-glycans lackfucose. In another embodiment, the N-glycans further comprise a corefucose.

In one embodiment, the invention comprises a method of enhancing animmune response in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of an Fc-containingpolypeptide comprising sialylated N-glycans, wherein the sialic acidresidues in the sialylated N-glycans contain α-2,3 linkages, and whereinat least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureselected from the group consisting ofSA(1-4)Gal(1-4)GlcNAc(1-4)Man(>=3)GlcNAc₂. In one embodiment, at least30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an oligosaccharide structure selectedfrom the group consisting of SA(1-3)Gal(1-3)GlcNAc(1-3)Man3GlcNAc2. Inany of the above embodiments, the SA could be NANA or NGNA, or an analogor derivative of NANA or NGNA. In one embodiment, the sialic acidresidues in the sialylated N-glycans are attached exclusively via α-2,3linkages.

In one embodiment, the invention comprises a method of treating aneoplastic disease (tumor) in a subject comprising administering to thesubject a therapeutically effective amount of an Fc-containingpolypeptide comprising sialylated N-glycans, wherein the sialic acidresidues in the sialylated N-glycans contain α-2,3 linkages, and whereinat least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureselected from the group consisting ofSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, the sialicacid residues in the sialylated N-glycans are attached exclusively viaα-2,3 linkages. In one embodiment, the subject has, or is at risk ofdeveloping, an infectious disease or a neoplastic disease. In oneembodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, at least 30%, 40%,50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, atleast 80% of the N-glycans on the Fc-containing polypeptide comprise anN-linked oligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In any of the above embodiments, the SA couldbe NANA or NGNA, or an analog or derivative of NANA or NGNA. In oneembodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide consisting of NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In oneembodiment, the N-glycans lack fucose. In another embodiment, theN-glycans further comprise a core fucose.

In any of the above identified embodiments, the Fc polypeptide can be anantibody or antibody fragment comprising sialylated N-glycans. In oneembodiment, the Fc polypeptide comprises N-glycans at a position thatcorresponds to the Asn297 site of a full-length heavy chain antibody,wherein the numbering is according to the EU index as in Kabat. In oneembodiment, the Fc polypeptide is an antibody or antibody fragmentcomprising or consisting essentially of SEQ ID NO:6 or SEQ ID NO:7. Inone embodiment the Fc-containing polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, plus or moremutations which result in an increased amount of sialic acid whencompared to the amount of sialic acid in the parent polypeptide. In oneembodiment the Fc-containing polypeptide comprises or consists of theamino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, plus one, two,three or four mutations which result in an increased amount of sialicacid when compared to the amount of sialic acid in the parentpolypeptide. In one embodiment, the parent polypeptide comprises theamino acid sequence of SEQ ID NO:6 or SEQ ID NO:7. In one embodiment,the Fc-containing polypeptide is an antibody or antibody fragmentcomprising a mutation at position 243 of the Fc region wherein thenumbering is according to EU index as in Kabat. In one embodiment, themutation is F243A. In one embodiment, the Fc-containing polypeptide isan antibody or antibody fragment comprising a mutation at position 264of the Fc region wherein the numbering is according to EU index as inKabat. In one embodiment, the mutation is V264A. In one embodiment, theFc-containing polypeptide is an antibody or antibody fragment comprisingmutations at positions 243 and 264 of the Fc region wherein thenumbering is according to EU index as in Kabat. In one embodiment, themutations are F243A and V264A.

In one embodiment the Fc-containing polypeptide has one or more of thefollowing properties when compared to a parent Fc-containingpolypeptide: increased effector function, increased ability to recruitimmune cells, and increased inflammatory properties.

The invention also comprises a method of enhancing an immune response ina subject in need thereof comprising: administering to the subject atherapeutically effective amount of an Fc-containing polypeptidecomprising N-glycans, wherein at at least 30%, 40%, 50%, 60%, 70%, 80%or 90% of the N-glycans on the Fc-containing polypeptide comprise anoligosaccharide structure selected from the group consisting ofSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂, In one embodiment, the sialicacid residues are exclusively attached through an α-2,3 linkage. In oneembodiment, the subject has, or is at risk of developing, an infectiousdisease or a neoplastic disease. In one embodiment, at least 30%, 40%,50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In oneembodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure consisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Inone embodiment, at least 80% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In any of the aboveembodiments, the SA could be NANA or NGNA, or an analog or derivative ofNANA or NGNA. In one embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%or 90% of the N-glycans on the Fc-containing polypeptide comprise anN-linked oligosaccharide structure consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, the N-glycans lackfucose. In another embodiment, the N-glycans further comprise a corefucose.

The invention also comprises a method of enhancing an immune response ina subject in need thereof comprising: administering to the subject atherapeutically effective amount of an Fc-containing polypeptidecomprising sialylated N-glycans, wherein the sialic acid residues in theFc-containing polypeptide contain an α-2,3 linkage, and wherein theFc-containing polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 6 or SEQ ID NO: 7, plus one or more mutationswhich result in an increased amount of sialic acid when compared to theamount of sialic acid in the parent polypeptide. In one embodiment, theFc-containing polypeptide comprises the amino acid sequence of SEQ IDNO:6 or SEQ ID NO:7, plus one, two, three or four mutations which resultin an increased amount of sialic acid when compared to the amount ofsilaic acid in the parent polypeptide. In one embodiment, the parentpolypeptide comprises the amino acid sequence of SEQ ID NO:6 or SEQ IDNO:7. In one embodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% ofthe N-glycans on the Fc-containing polypeptide comprise anoligosaccharide structure selected from the group consisting ofSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, at least 30%,40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, atleast 80% of the N-glycans on the Fc-containing polypeptide comprise anN-linked oligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 30%, 40%, 50%,60%, 70%, 80% or 90% of the N-glycans on the Fc-containing polypeptidecomprise an N-linked oligosaccharide structure selected from the groupconsisting of NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, the sialicacid residues in the sialylated N-glycans are attached exclusively viaα-2,3 linkages.

The invention also comprises a pharmaceutical formulation comprising anFc-containing polypeptide, wherein the Fc-containing polypeptidecomprises sialylated N-glycans, wherein the sialic acid residues in thesialylated N-glycans are attached exclusively via α-2,3 linkages.

The invention also comprises a pharmaceutical formulation comprising anFc-containing polypeptide, wherein the Fc-containing polypeptidecomprises sialylated N-glycans, wherein the sialic acid residues in thesialylated N-glycans contain α-2,3 linkages, and wherein at least 30%,40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In oneembodiment, at least wherein at least 30%, 40%, 50%, 60%, 70%, 80% or90% of the N-glycans on the Fc-containing polypeptide comprise anN-linked oligosaccharide structure selected from the group consisting ofSA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, at least whereinat least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureconsisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 80%of the N-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least wherein at least30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureconsisting of NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, the sialicacid residues in the sialylatd N-glycans are attached exclusively viaα-2,3 linkages. In one embodiment, the N-glycans lack fucose. In anotherembodiment, the N-glycans further comprise a core fucose.

In any one of the embodiments directed to pharmaceutical formulations,the Fc-containing polypeptide can be an antibody or an antibody fragmentcomprising sialylated N-glycans. In one embodiment, the Fc polypeptidecomprises N-glycans at a position that corresponds to the Asn297 site ofa full-length heavy chain antibody, wherein the numbering is accordingto the EU index as in Kabat. In one embodiment, the Fc-containingpolypeptide is an antibody or antibody fragment comprising the aminoacid sequence of SEQ ID NO:6 or SEQ ID NO:7, plus one or more mutationswhich result in an increased amount of sialic acid when compared to theamount of silaic acid in the parent polypeptide. In one embodiment, theFc-containing polypeptide is an antibody or antibody fragment comprisingor consisting essentially of the amino acid sequence of SEQ ID NO: 6 orSEQ ID NO: 7, plus one, two, three or four mutations which result in anincreased amount of sialic acid when compared to the amount of sialicacid in the parent polypeptide. In one embodiment, the parentpolypeptide comprises the amino acid sequence of SEQ ID NO:6 or SEQ IDNO:7. In one embodiment, the Fc-containing polypeptide is an antibody orantibody fragment comprising mutations at positions 243 and 264 of theFc region wherein the numbering is according to EU index as in Kabat. Inone embodiment, the mutations are F243A and V264A. In one embodiment theFc-containing polypeptide has one or more of the following propertieswhen compared to a parent Fc-containing polypeptide: increased effectorfunction, increased ability to recruit immune cells, and increasedinflammatory properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the anti-tumor efficacy (by reduction in tumor volume) ofvarious antibodies in a 4T1-Luc2 model.

FIG. 2 shows the anti-tumor efficacy (by reduction in tumor volume) ofvarious antibodies in a 4T1-Luc2 model.

FIG. 3 shows the tumor growth inhibition (TGI) of various antibodies ina 4T1-Luc2 model.

FIG. 4 shows images of cancer metastasis to lung tissue fromtumor-implanted mice treated with various antibodies.

FIG. 5 shows the effect of alpha2,3 sialylated Fc in an AIA model asdescribed in Example 4.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “G0” when used herein refers to a complex bi-antennaryoligosaccharide without galactose or fucose, GlcNAc₂Man₃GlcNAc₂.

The term “G1” when used herein refers to a complex bi-antennaryoligosaccharide without fucose and containing one galactosyl residue,GalGlcNAc₂Man₃GlcNAc₂.

The term “G2” when used herein refers to a complex bi-antennaryoligosaccharide without fucose and containing two galactosyl residues,Gal₂GlcNAc₂Man₃GlcNAc₂.

The term “G0F” when used herein refers to a complex bi-antennaryoligosaccharide containing a core fucose and without galactose,GlcNAc₂Man₃GlcNAc₂F.

The term “G1F” when used herein refers to a complex bi-antennaryoligosaccharide containing a core fucose and one galactosyl residue,GalGlcNAc₂Man₃GlcNAc₂F.

The term “G2F” when used herein refers to a complex bi-antennaryoligosaccharide containing a core fucose and two galactosyl residues,Gal₂GlcNAc₂Man₃GlcNAc₂F.

The term “Man5” when used herein refers to the oligosaccharide structureshown as

The term “GFI 5.0” when used herein refers to glycoengineered Pichiapastoris strains that produce glycoproteins having predominantlyGal₂GlcNAc₂Man₃GlcNAc₂ N-glycans.

The term “GFI 6.0” when used herein refers to glycoengineered Pichiapastoris strains that produce glycoproteins having predominantlySA₂Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans.

The term “GS5.0”, when used herein refers to the N-glycosylationstructure Gal₂GlcNAc₂Man₃GlcNAc₂.

The term “GS5.5”, when used herein refers to the N-glycosylationstructure SAGal₂GlcNAc₂Man₃GlcNAc₂, which when produced in Pichiapastoris strains to which α-2,6 sialyl transferase has beenglycoengineered result in α-2,6-linked sialic acid, which when producedin Pichia pastoris strains to which α-2,3 sialyl transferase has beenglycoengineered result in α-2,3-linked sialic acid, and which whenproduced in Pichia pastoris strains to which α-2,6 sialyl transferaseand α-2,3 sialyl transferase have been glycoengineered result in amixture of α-2,6- and α-2,3-linked sialic acid species. The sialic acidproduced in Pichia pastoris is of the N-acetyl neuraminic acid (NANA)type unless the strain has been engineered to express CMP-NANAhydroxylase wherein the sialic acid will be a mixture of N-glycolylneuraminic acid (NGNA) and NANA.

The term “GS6.0”, when used herein refers to the N-glycosylationstructure SA₂Gal₂GlcNAc₂Man₃GlcNAc₂, which when produced in Pichiapastoris strains to which α-2,6 sialyl transferase has beenglycoengineered result in α-2,6-linked sialic acid and which whenproduced in Pichia pastoris strains to which α-2,3 sialyl transferasehas been glycoengineered result in α-2,3-linked sialic acid, and whichwhen produced in Pichia pastoris strains to which α-2,6 sialyltransferase and α-2,3 sialyl transferase have been glycoengineeredresult in a mixture of α-2,6- and α-2,3-linked sialic acid species. Thesialic acid produced in Pichia pastoris is of the N-acetyl neuraminicacid (NANA) type unless the strain has been engineered to expressCMP-NANA hydroxylase wherein the sialic acid will be a mixture ofN-glycolyl neuraminic acid (NGNA) and NANA.

The term “wild type” or “wt” when used herein in connection to a Pichiapastoris strain refers to a native Pichia pastoris strain that has notbeen subjected to genetic modification to control glycosylation.

The term “antibody”, when used herein refers to an immunoglobulinmolecule capable of binding to a specific antigen through at least oneantigen recognition site located in the variable region of theimmunoglobulin molecule. As used herein, the term encompasses not onlyintact polyclonal or monoclonal antibodies, consisting of fourpolypeptide chains, i.e. two identical pairs of polypeptide chains, eachpair having one “light” chain (LC) (about 25 kDa) and one “heavy” chain(HC) (about 50-70 kDa), but also fragments thereof, such as Fab, Fab′,F(ab′)₂, Fv, single chain (ScFv), mutants thereof, bispecific formats,fusion proteins comprising an antibody portion, and any other modifiedconfiguration of an immunoglobulin molecule that comprises an antigenrecognition site and at least the portion of the C_(H)2 domain of theheavy chain immunoglobulin constant region which comprises an N-linkedglycosylation site of the C_(H)2 domain, or a variant thereof. As usedherein the term includes an antibody of any class, such as IgG (forexample, IgG1, IgG2, IgG3 or IgG4), IgM, IgA, IgD and IgE, respectively.

The term “consensus sequence of C_(H)2” when used herein refers to theamino acid sequence of the C_(H)2 domain of the heavy chain constantregion containing an N-linked glycosylation site which was derived fromthe most common amino acid sequences found in C_(H)2 domains from avariety of antibodies.

The term “Fc region” is used to define a C-terminal, or so-calledeffector region, of an immunoglobulin heavy chain. The “Fc region” maybe a native sequence Fc region or a variant Fc region. Although theboundaries of the Fc region of an immunoglobulin heavy chain might vary,the human IgG heavy chain Fc region is usually defined to stretch froman amino acid residue at position Cys226, or from Pro230, to thecarboxyl-terminus thereof. The Fc region of an immunoglobulin comprisestwo constant domains, CH2 and CH3, and can optionally comprise a hingeregion. In one embodiment, the Fc region comprises the amino acidsequence of SEQ ID NO:6. In one embodiment, the Fc region comprises theamino acid sequence of SEQ ID NO:7. In another embodiment, the Fc regioncomprises the amino acid sequence of SEQ ID NO:6, with the addition of alysine (K) residue at the 3′ end. The Fc region contains a singleN-linked glycosylation site in the CH2 domain that corresponds to theAsn297 site of a full-length heavy chain of an antibody, wherein thenumbering is according to the EU index as in Kabat.

The term “Fc-containing polypeptide” refers to a polypeptide, such as anantibody or immunoadhesin, which comprises an Fc region or a fragment ofan Fc region which retains the N-linked glycosylation site in the CH2domain and retains the ability to recruit immune cells. This termencompasses polypeptides comprising or consisting of (or consistingessentially of) an Fc region either as a monomer or dimeric species.Polypeptides comprising an Fc region can be generated by papaindigestion of antibodies or by recombinant DNA technology.

The term “parent antibody”, “parent immunoglobulin” or “parentFc-containing polypeptide” when used herein refers to an antibody orFc-containing polypeptide which lacks the Fc region mutations disclosedherein. A parent Fc-containing polypeptide may comprise a nativesequence Fc region or an Fc region with pre-existing amino acid sequencemodifications. A native sequence Fc region comprises an amino acidsequence identical to the amino acid sequence of an Fc region found innature. Native sequence Fc regions include the native sequence humanIgG1 Fc region, the native sequence human IgG2 Fc region, the nativesequence human IgG3 Fc region and the native sequence human IgG4 Fcregion as well as naturally occurring variants thereof. When used as acomparator, a parent antibody or a parent Fc-containing polypeptide canbe expressed in any cell. In one embodiment, the parent antibody or aparent Fc-containing polypeptide is expressed in the same cell as theFc-containing polypeptide of the invention.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the “binding domain” of a heterologous “adhesin”protein (e.g. a receptor, ligand or enzyme) with an immunoglobulinconstant domain. Structurally, the immunoadhesins comprise a fusion ofthe adhesin amino acid sequence with the desired binding specificitywhich is other than the antigen recognition and binding site (antigencombining site) of an antibody (i.e. is “heterologous”) and animmunoglobulin constant domain sequence. The term “ligand bindingdomain” as used herein refers to any native cell-surface receptor or anyregion or derivative thereof retaining at least a qualitative ligandbinding ability of a corresponding native receptor. In a specificembodiment, the receptor is from a cell-surface polypeptide having anextracellular domain that is homologous to a member of theimmunoglobulin supergenefamily. Other receptors, which are not membersof the immunoglobulin supergenefamily but are nonetheless specificallycovered by this definition, are receptors for cytokines, and inparticular receptors with tyrosine kinase activity (receptor tyrosinekinases), members of the hematopoietin and nerve growth factor whichpredispose the mammal to the disorder in question. In one embodiment,the disorder is cancer. Methods of making immunoadhesins are well knownin the art. See, e.g., WO00/42072.

The term “Fc mutein antibody” when used herein refers to an antibodycomprising one or more mutations in the Fc region.

The term “Fc mutein” when used herein refers to an Fc-containingpolypeptide in which one or more point mutations have been made to theFc region.

The term “Fc mutation” when used herein refers to a mutation made to theFc region of an Fc-containing polypeptide. Examples of such a mutationinclude the F243A or V264A mutations (wherein the numbering is accordingto EU index as in Kabat). For example, the term “F243A” refers to amutation from F (wild-type) to A at position 243 of the Fc region of anFc-containing polypeptide. The term “V264A” refers to a mutation from V(wild-type) to A at position 264 of the Fc region of an Fc-containingpolypeptide. The position 243 and 264 represent the amino acid positionsin the CH2 domain of the Fc region of an Fc-containing polypeptide. Theterm “double Fc mutein” when used herein refers to an Fc-containingpolypeptide comprising mutations F243A and V264A.

Throughout the present specification and claims, the numbering of theresidues in an immunoglobulin heavy chain or an Fc-containingpolypeptide is that of the EU index as in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), expresslyincorporated herein by reference. The “EU index as in Kabat” refers tothe residue numbering of the human IgG1 EU antibody.

The term “effector function” as used herein refers to a biochemicalevent that results from the interaction of an antibody Fc region with anFc receptor or ligand. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependentcellular phagocytosis (ADCP); phagocytosis; down regulation of cellsurface receptors (e. g. B cell receptor; BCR), etc. Such effectorfunctions can be assessed using various assays known in the art.

The term “glycoengineered Pichia pastoris” when used herein refers to astrain of Pichia pastoris that has been genetically altered to expresshuman-like N-glycans. For example, the GFI 5.0, GFI 5.5 and GFI 6.0strains described above.

The terms “N-glycan”, “glycoprotein” and “glycoform” when used hereinrefer to an N-linked oligosaccharide, e.g., one that is attached by anasparagine-N-acetylglucosamine linkage to an asparagine residue of apolypeptide. Predominant sugars found on glycoproteins are galactose,mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine(GlcNAc) and sialic acid (Sia or SA, including NANA, NGNA andderivatives and analogs thereof, including acetylated NANA or acetylatedNGNA). In glycoengineered Pichia pastoris, sialic acid is exclusivelyN-acetyl-neuraminic acid (NANA) (Hamilton et al., Science 313 (5792):1441-1443 (2006)) unless the strains are further engineered to expressCMP-NANA hydroxylase to convert NANA into NGNA. N-glycans have a commonpentasaccharide core of Man₃GlcNAc₂, wherein “Man” refers to mannose,“Glc” refers to glucose, “NAc” refers to N-acetyl, and GlcNAc refers toN-acetylglucosamine. N-glycans differ with respect to the number ofbranches (antennae) comprising peripheral sugars (e.g., GlcNAc,galactose, fucose and sialic acid) that are added to the Man₃GlcNAc₂(“Man3”) core structure which is also referred to as the “trimannosecore”, the “pentasaccharide core” or the “paucimannose core”. N-glycansare classified according to their branched constituents (e.g., highmannose, complex or hybrid).

As used herein, the term “sialic acid” or “SA” or “Sia” refers to anymember of the sialic acid family, including without limitation:N-acetylneuraminic acid (Neu5Ac or NANA), N-glycolylneuraminic acid(NGNA) and any analog or derivative thereof (including those arisingfrom acetylation at any position on the sialic acid molecule). Sialicacid is a generic name for a group of about 30 naturally occurringacidic carbohydrates that are essential components of a large number ofglycoconjugates. Schauer, Biochem. Society Transactions, 11, 270-271(1983). Sialic acids typically reside at the nonreducing, or terminal,end of oligosaccharides. In humans, sialic acids are usually theterminal residue of the oligosaccharides. N-acetylneuraminic acid (NANA)is the most common sialic acid form and N-glycolylneuraminic acid (NGNA)is the second most common form. Schauer, Glycobiology, 1, 449-452(1991). NGNA is widespread throughout the animal kingdom and, accordingto species and tissue, often constitutes a significant proportion of theglycoconjugate-bound sialic acid. Certain species such as chicken andman are exceptional, since they lack NGNA in normal tissues. Corfield,et al., Cell Biology Monographs, 10, 5-50 (1982). In human serumsamples, the percentage of sialic acid in the form of NGNA is reportedto be 0.01% of the total sialic acid. Schauer, “Sialic Acids asAntigenic Determinants of Complex Carbohydrates”, found in The MolecularImmunology of Complex Carbohydrates, (Plenum Press, New York, 1988).

The term “human-like N-glycan”, as used herein, refers to N-linkedoligosaccharides which closely resemble the oligosaccharides produced bynon-engineered, wild-type human cells. For example, wild-type Pichiapastoris and other lower eukaryotic cells typically producehypermannosylated proteins at N-glycosylation sites. The host cellsdescribed herein produce glycoproteins (for example, antibodies)comprising human-like N-glycans that are not hypermannosylated. In someembodiments, the host cells of the present invention are capable ofproducing human-like N-glycans with hybrid and/or complex N-glycans. Thespecific type of “human-like” glycans present on a specific glycoproteinproduced from a host cell of the invention will depend upon the specificglycoengineering steps that are performed in the host cell.

The term “high mannose” type N-glycan when used herein refers to anN-glycan having five or more mannose residues.

The term “complex” type N-glycan when used herein refers to an N-glycanhaving at least one GlcNAc attached to the 1,3 mannose arm and at leastone GlcNAc attached to the 1,6 mannose arm of a “trimannose” core.Complex N-glycans may also have galactose (“Gal”) orN-acetylgalactosamine (“GalNAc”) residues that are optionally modifiedwith sialic acid or derivatives (e.g., “NANA” or “NeuAc”, where “Neu”refers to neuraminic acid and “Ac” refers to acetyl). Complex N-glycansmay also have intrachain substitutions comprising “bisecting” GlcNAc andcore fucose (“Fuc”). As an example, when a N-glycan comprises abisecting GlcNAc on the trimannose core, the structure can berepresented as Man₃GlcNAc₂(GlcNAc) or Man₃GlcNAc₃. When an N-glycancomprises a core fucose attached to the trimannose core, the structuremay be represented as Man₃GlcNAc₂(Fuc). Complex N-glycans may also havemultiple antennae on the “trimannose core,” often referred to as“multiple antennary glycans.”

The term “hybrid” N-glycan when used herein refers to an N-glycan havingat least one GlcNAc on the nonreducing terminus of the 1,3 mannose armof the trimannose core and zero or more than one additional mannose onthe nonreducing terminus of the 1,6 mannose arm of the trimannose core.

When referring to “mole percent” of a glycan present in a preparation ofa glycoprotein, the term means the molar percent of a particular glycanpresent in the pool of N-linked oligosaccharides released when theprotein preparation is treated with PNGase and then quantified by amethod that is not affected by glycoform composition, (for instance,labeling a PNGase released glycan pool with a fluorescent label such as2-aminobenzamide and then separating by high performance liquidchromatography or capillary electrophoresis and then quantifying glycansby fluorescence intensity). For example, 50 mole percent NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ means that 50 percent of the released glycans areNANA₂ Gal₂GlcNAc₂Man₃GlcNAc₂ and the remaining 50 percent are comprisedof other N-linked oligosaccharides.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity of the protein. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity(see, e.g., Watson et al. (1987) Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,substitutions of structurally or functionally similar amino acids areless likely to disrupt biological activity. Exemplary conservativesubstitutions are listed below:

Original residue Conservative substitution Ala (A) Gly; Ser Arg (R) Lys;His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu(E) Asp; Gln Gly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile;Val Lys (K) Arg; His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P)Ala Ser (S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V)Ile; Leu

Glycosylation of immunoglobulin G (IgG) in the Fc region, Asn297(according to the EU numbering system), has been shown to be arequirement for optimal recognition and activation of effector pathwaysincluding antibody dependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC), Wright & Morrison, Trends inBiotechnology, 15: 26-31 (1997), Tao & Morrison, J. Immunol.,143(8):2595-2601 (1989). As such, glycosylation engineering in theconstant region of IgG has become an area of active research for thedevelopment of therapeutic monoclonal antibodies (mAbs). It has beenestablished that the presence of N-linked glycosylation at Asn297 iscritical for mAb activity in immune effector function assays includingADCC, Rothman (1989), Lifely et al., Glycobiology, 5:813-822 (1995),Umana (1999), Shields (2002), and Shinkawa (2003), and complementdependent cytotoxicity (CDC), Hodoniczky et al., Biotechnol. Prog.,21(6): 1644-1652 (2005), and Jefferis et al., Chem. Immunol., 65:111-128 (1997). This effect on function has been attributed to thespecific conformation adopted by the glycosylated Fc domain, whichappears to be lacking when glycosylation is absent. More specifically,IgG which lacks glycosylation in the Fc C_(H)2 domain does not bind toFcγR, including FcγRI, FcγRII, and FcγRIII, Rothman (1989).

Not only does the presence of glycosylation appear to play a role in theeffector function of an antibody, the particular composition of theN-linked oligosaccharide is also important. For example, the presence offucose shows a marked effect on in vitro FcγRIIIa binding and in vitroADCC, Rothman (1989), and Li et al., Nat. Biotechnol. 24(2): 2100-215(2006). Recombinant antibodies produced by mammalian cell culture, suchas CHO or NSO, contain N-linked oligosaccharides that are predominatelyfucosylated, Hossler et al., Biotechnology and Bioengineering,95(5):946-960 (2006), Umana (1999), and Jefferis et al., Biotechnol.Prog. 21:11-16 (2005). Additionally, there is evidence that sialylationin the Fc region may impart anti-inflammatory properties to antibodies.Intravenous immunoglobulin (WIG) purified over a lectin column to enrichfor the sialylated form showed a distinct anti-inflammatory effectlimited to the sialylated Fc fragment and was linked to an increase inexpression of the inhibitory receptor FcγRIIb, Nimmerjahn and Ravetch.,J. Exp. Med. 204:11-15 (2007).

Glycosylation in the Fc region of an antibody derived from mammaliancell lines typically consists of a heterogeneous mix of glycoforms, withthe predominant forms typically being comprised of the complexfucosylated glycoforms: G0F, G1F, and, to a lesser extent, G2F. Possibleconditions resulting in incomplete galactose transfer to the G0Fstructure include, but are not limited to, non-optimized galactosetransfer machinery, such as β-1,4 galactosyl transferase, and poorUDP-galactose transport into the Golgi apparatus, suboptimal cellculture and protein expression conditions, and steric hindrance by aminoacid residues neighboring the oligosaccharide. While each of theseconditions may modulate the ultimate degree of terminal galactose, it isthought that subsequent sialic acid transfer to the Fc oligosaccharideis inhibited by the closed pocket configuration of the C_(H)2 domain.See, for example, FIG. 1, Jefferis, R., Nature Biotech., 24 (10):1230-1231, 2006. Without the correct terminal monosaccharide,specifically galactose, or with insufficient terminal galactosylatedforms, there is little possibility of producing a sialylated form,capable of acting as a therapeutic protein, even when produced in thepresence of sialyl transferase. Protein engineering and structuralanalysis of human IgG-Fc glycoforms has shown that glycosylationprofiles are affected by Fc conformation, such as the finding thatincreased levels of galactose and sialic acid on oligosaccharidesderived from CHO-produced IgG3 could be achieved when specific singleamino acid mutations in the Fc pocket were mutated, to an alanineincluding F241, F243, V264, D265 and R301. Lund et al., J. Immunol.157(11); 4963-4969 (1996). It was further shown that certain mutationshad some effect on cell-mediated superoxide generation and complementmediated red cell lysis, which are used as surrogate markers for FcγRIand C1q binding, respectively.

Yeast have been genetically engineered to produce host strains capableof secreting glycoproteins with highly uniform glycosylation. Choi etal., PNAS, USA 100(9): 5022-5027 (2003) describes the use of librariesof a 1,2 mannosidase catalytic domains andN-acetylglucosaminyltransferase I catalytic domains in combination witha library of fungal type II membrane protein leader sequences tolocalize the catalytic domains to the secretory pathway. In this way,strains were isolated that produced in vivo glycoproteins with uniformMan₅GlcNAc₂ or GlcNAcMan5GlcNAc₂ N-glycan structures. Hamilton et al.,Science 313 (5792): 1441-1443 (2006) described the production of aglycoprotein, erythropoietin, produced in Pichia pastoris, as having aglycan composition that consisted predominantly of a bisialylated glycanstructure, GS6.0, NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ (90.5%) andmonosialylated, GS5.5, NANAGal₂GlcNAc₂ Man₃GlcNAc₂ (7.9%). However, anantibody produced in a similar strain will have a markedly lower contentof sialylated N-glycans due to the relatively low level of terminalgalactose substrate in the antibody. It has also recently been shownthat sialylation of a Fc oligosaccharide imparts anti-inflammatoryproperties on therapeutic intravenous gamma globulin and its Fcfragments, Kaneko et al., Science 313(5787): 670-673 (2006), and thatthe anti-inflammatory activity is dependent on the a 2,6-linked but notthe α-2,3 linked, form of sialic acid, Anthony et al., Science, 320:373-376 (2008).

As used herein, the term “neoplastic disease” includes any diseaseresulting from an abnormal, uncontrolled growth of cells. Neoplasms maybe benign, pre-malignant (carcinoma in situ) or malignant (cancer) withor without metastasis or metastatic potential.

As used herein, the term “infectious disease” includes any conditioncaused by a microorganism or other agent, such as a bacterium, fungus,or virus that enters the body of an organism.

Host Organisms and Cell Lines

The Fc-containing polypeptides of this invention can be made in any hostorganism, cell line or in silico. In one embodiment, an Fc-containingpolypeptide of the invention is made in a host cell which is capable ofproducing sialylated N-glycans.

In one embodiment, an Fc-containing polypeptide of the invention is madein a mammalian cell where the cell either endogenously or throughgenetic or process manipulation produces glycoproteins containing onlyterminal α-2,3 sialic acid. The propagation of mammalian cells inculture (tissue culture) has become a routine procedure. Examples ofuseful mammalian host cell lines are monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture); baby hamster kidneycells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mousesertoli cells (TM4,); monkey kidney cells (CV1 ATCC CCL 70); Africangreen monkey kidney cells (VERO-76, ATCC CRL-1587); human cervicalcarcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammarytumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells;hybridoma cell lines; NSO; SP2/0; and a human hepatoma line (Hep G2).

In one embodiment, an Fc-containing polypeptide of the invention can bemade in a plant cell which is engineered to produce α-2,3 sialylatedN-glycans. See, e.g., Cox et al., Nature Biotechnology (2006) 24,1591-1597 (2006) and Castilho et al., J. Biol. Chem. 285(21):15923-15930 (2010).

In one embodiment, an Fc-containing polypeptide of the invention can bemade in an insect cell which is engineered to produce α-2,3 sialylatedN-glycans. See, e.g., Harrison and Jarvis, Adv. Virus Res. 68:159-91(2006).

In one embodiment, an Fc-containing polypeptide of the invention can bemade in a bacterial cell which is engineered to produce α-2,3 sialylatedN-glycans. See, e.g., Lizak et al., Bioconjugate Chem. 22:488-496(2011).

In one embodiment, an Fc-containing polypeptide of the invention can bemade in a lower eukaryotic host cell or organism. Recent developmentsallow for the production of fully humanized therapeutics in lowereukaryotic host organisms, yeast and filamentous fungi, such as Pichiapastoris, Gerngross et al., U.S. Pat. No. 7,029,872 and U.S. Pat. No.7,449,308, the disclosures of which are hereby incorporated byreference. See also Jacobs et al., Nature Protocols 4(1):58-70 (2009).Applicants herein have further developed modified Pichia pastoris hostorganisms and cell lines capable of expressing antibodies comprising twomutations to the amino acids at positions 243 and 264 in the Fc regionof the heavy chain. The antibodies having these mutations had increasedlevels and a more homogeneous composition of the α-2,3 linked sialylatedN-glycans when compared to a parent antibody.

In one embodiment, an Fc-containing polypeptide of the invention is madein a host cell, more preferably a yeast or filamentous fungal host cell,that has been engineered to produce glycoproteins having a predominantN-glycan comprising a terminal α-2,3-sialic acid. In one embodiment ofthe invention, the predominant N-glycan is the α-2,3 linked form ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂, produced in strains glycoengineered withα-2,3 sialyl transferase which do not produce any α-2,6 linked sialicacid.

The cell lines to be used to make the Fc-containing polypeptides of theinvention can be any cell line, in particular cell lines with thecapability of producing one or more α-2,3-sialylated glycoproteins.Those of ordinary skill in the art would recognize and appreciate thatthe materials and methods described herein are not limited to thespecific strain of Pichia pastoris provided as an example herein, butcould include any Pichia pastoris strain or other yeast or filamentousfungal strains in which N-glycans with one or more terminal galactose,such as Gal₂GlcNAc₂Man₃, are produced. The terminal galactose acts as asubstrate for the production of α-2,3-linked sialic acid, resulting inthe N-glycan structure SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Examples of suitablestrains are described in U.S. Pat. No. 7,029,872, U.S. Publication No.2006-0286637 and Hamilton et al., Science 313 (5792): 1441-1443 (2006),the descriptions of which are incorporated herein as if set forth atlength.

In general, lower eukaryotes such as yeast are used for expression ofthe proteins, particularly glycoproteins because they can beeconomically cultured, give high yields, and when appropriately modifiedare capable of suitable glycosylation. Yeast particularly offersestablished genetics allowing for rapid transformations, tested proteinlocalization strategies and facile gene knock-out techniques. Suitablevectors have expression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

While the invention has been demonstrated herein using themethylotrophic yeast Pichia pastoris, other useful lower eukaryote hostcells include Pichia pastoris, Pichia finlandica, Pichia trehalophila,Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta,Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporiumi lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Yarrowia lipotylica and Neurosporacrassa. Various yeasts, such as K. lactis, Pichia pastoris, Pichiamethanolica, Yarrowia lipolytica and Hansenula polymorpha areparticularly suitable for cell culture because they are able to grow tohigh cell densities and secrete large quantities of recombinant protein.Likewise, filamentous fungi, such as Aspergillus niger, Fusarium sp,Neurospora crassa and others can be used to produce glycoproteins of theinvention at an industrial scale.

Lower eukaryotes, particularly yeast and filamentous fungi, can begenetically modified so that they express glycoproteins in which theglycosylation pattern is human-like or humanized. As indicated above,the term “human-like N-glycan”, as used herein refers, to the N-linkedoligosaccharides which closely resemble the oligosaccharides produced bynon-engineered, wild-type human cells. In preferred embodiments of thepresent invention, the host cells of the present invention are capableof producing human-like glycoproteins with hybrid and/or complexN-glycans; i.e., “human-like N-glycosylation.” The specific “human-like”glycans predominantly present on glycoproteins produced from the hostcells of the invention will depend upon the specific engineering stepsthat are performed. In this manner, glycoprotein compositions can beproduced in which a specific desired glycoform is predominant in thecomposition. Such can be achieved by eliminating selected endogenousglycosylation enzymes and/or genetically engineering the host cellsand/or supplying exogenous enzymes to mimic all or part of the mammalianglycosylation pathway as described in U.S. Pat. No. 7,449,308. Ifdesired, additional genetic engineering of the glycosylation can beperformed, such that the glycoprotein can be produced with or withoutcore fucosylation. Use of lower eukaryotic host cells is furtheradvantageous in that these cells are able to produce highly homogenouscompositions of glycoprotein, such that the predominant glycoform of theglycoprotein may be present as greater than thirty mole percent of theglycoprotein in the composition. In particular aspects, the predominantglycoform may be present in greater than forty mole percent, fifty molepercent, sixty mole percent, seventy mole percent and, most preferably,greater than eighty mole percent of the glycoprotein present in thecomposition.

Lower eukaryotes, particularly yeast, can be genetically modified sothat they express glycoproteins in which the glycosylation pattern ishuman-like or humanized. Such can be achieved by eliminating selectedendogenous glycosylation enzymes and/or supplying exogenous enzymes asdescribed by Gerngross et al., U.S. Pat. No. 7,449,308. For example, ahost cell can be selected or engineered to be depleted in α1,6-mannosyltransferase activities, which would otherwise add mannose residues ontothe N-glycan on a glycoprotein.

In one embodiment, the host cell further includes an α1,2-mannosidasecatalytic domain fused to a cellular targeting signal peptide notnormally associated with the catalytic domain and selected to target theα1,2-mannosidase activity to the ER or Golgi apparatus of the host cell.Passage of a recombinant glycoprotein through the ER or Golgi apparatusof the host cell produces a recombinant glycoprotein comprising aMan₅GlcNAc₂ glycoform, for example, a recombinant glycoproteincomposition comprising predominantly a Man₅GlcNAc₂ glycoform. Forexample, U.S. Pat. Nos. 7,029,872 and 7,449,308 and U.S. PublishedPatent Application No. 2005/0170452 disclose lower eukaryote host cellscapable of producing a glycoprotein comprising a Man₅GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a GlcNAc transferase I (GnT I) catalytic domain fused to acellular targeting signal peptide not normally associated with thecatalytic domain and selected to target GlcNAc transferase I activity tothe ER or Golgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising a GlcNAcMan₅GlcNAc₂ glycoform, forexample a recombinant glycoprotein composition comprising predominantlya GlcNAcMan₅GlcNAc₂ glycoform. U.S. Pat. Nos. 7,029,872 and 7,449,308and U.S. Published Patent Application No. 2005/0170452 disclose lowereukaryote host cells capable of producing a glycoprotein comprising aGlcNAcMan₅GlcNAc₂ glycoform. The glycoprotein produced in the abovecells can be treated in vitro with a hexosaminidase to produce arecombinant glycoprotein comprising a Man₅GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a mannosidase II catalytic domain fused to a cellular targetingsignal peptide not normally associated with the catalytic domain andselected to target mannosidase II activity to the ER or Golgi apparatusof the host cell. Passage of the recombinant glycoprotein through the ERor Golgi apparatus of the host cell produces a recombinant glycoproteincomprising a GlcNAcMan₃GlcNAc₂ glycoform, for example a recombinantglycoprotein composition comprising predominantly a GlcNAcMan₃GlcNAc₂glycoform. U.S. Pat. No. 7,029,872 and U.S. Published Patent ApplicationNo. 2004/0230042 discloses lower eukaryote host cells that expressmannosidase II enzymes and are capable of producing glycoproteins havingpredominantly a GlcNAcMan₃GlcNAc₂ glycoform. The glycoprotein producedin the above cells can be treated in vitro with a hexosaminidase toproduce a recombinant glycoprotein comprising a Man₃GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes GlcNAc transferase II (GnT II) catalytic domain fused to acellular targeting signal peptide not normally associated with thecatalytic domain and selected to target GlcNAc transferase II activityto the ER or Golgi apparatus of the host cell. Passage of therecombinant glycoprotein through the ER or Golgi apparatus of the hostcell produces a recombinant glycoprotein comprising a GlcNAc₂Man₃GlcNAc₂glycoform, for example a recombinant glycoprotein composition comprisingpredominantly a GlcNAc₂Man₃GlcNAc₂ glycoform. U.S. Pat. Nos. 7,029,872and 7,449,308 and U.S. Published Patent Application No. 2005/0170452disclose lower eukaryote host cells capable of producing a glycoproteincomprising a GlcNAc₂Man₃GlcNAc₂ glycoform. The glycoprotein produced inthe above cells can be treated in vitro with a hexosaminidase to producea recombinant glycoprotein comprising a Man₃GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a galactosyltransferase catalytic domain fused to a cellulartargeting signal peptide not normally associated with the catalyticdomain and selected to target galactosyltransferase activity to the ERor Golgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising a GalGlcNAc₂ Man₃GlcNAc₂ orGal₂GlcNAc₂Man₃GlcNAc₂ glycoform, or mixture thereof for example arecombinant glycoprotein composition comprising predominantly aGalGlcNAc₂Man₃GlcNAc₂ glycoform or Gal₂GlcNAc₂Man₃GlcNAc₂ glycoform ormixture thereof. U.S. Pat. No. 7,029,872 and U.S. Published PatentApplication No. 2006/0040353 discloses lower eukaryote host cellscapable of producing a glycoprotein comprising a Gal₂GlcNAc₂ Man₃GlcNAc₂glycoform. The glycoprotein produced in the above cells can be treatedin vitro with a galactosidase to produce a recombinant glycoproteincomprising a GlcNAc₂Man₃ GlcNAc₂ glycoform, for example a recombinantglycoprotein composition comprising predominantly a GlcNAc₂Man₃GlcNAc₂glycoform.

In a further embodiment, the immediately preceding host cell furtherincludes a sialyltransferase catalytic domain fused to a cellulartargeting signal peptide not normally associated with the catalyticdomain and selected to target sialyltransferase activity to the ER orGolgi apparatus of the host cell. In a preferred embodiment, thesialyltransferase is an α-2,3-sialyltransferase. Passage of therecombinant glycoprotein through the ER or Golgi apparatus of the hostcell produces a recombinant glycoprotein comprising predominantly aNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂ glycoform or NANAGal₂GlcNAc₂Man₃GlcNAc₂glycoform or mixture thereof. For lower eukaryote host cells such asyeast and filamentous fungi, it is useful that the host cell furtherinclude a means for providing CMP-sialic acid for transfer to theN-glycan. U.S. Published Patent Application No. 2005/0260729 discloses amethod for genetically engineering lower eukaryotes to have a CMP-sialicacid synthesis pathway and U.S. Published Patent Application No.2006/0286637 discloses a method for genetically engineering lowereukaryotes to produce sialylated glycoproteins. To enhance the amount ofsialylation, it can be advantageous to construct the host cell toinclude two or more copies of the CMP-sialic acid synthesis pathway ortwo or more copies of the sialylatransferase. The glycoprotein producedin the above cells can be treated in vitro with a neuraminidase toproduce a recombinant glycoprotein comprising predominantly aGal₂GlcNAc₂Man₃GlcNAc₂ glycoform or GalGlcNAc₂Man₃GlcNAc₂ glycoform ormixture thereof.

Any one of the preceding host cells can further include one or moreGlcNAc transferase selected from the group consisting of GnT III, GnTIV, GnT V, GnT VI, and GnT IX to produce glycoproteins having bisected(GnT III) and/or multiantennary (GnT IV, V, VI, and IX) N-glycanstructures such as disclosed in U.S. Published Patent Application Nos.2005/0208617 and 2007/0037248. Further, the proceeding host cells canproduce recombinant glycoproteins (for example, antibodies) comprisingSA(1-4)Gal(1-4)GlcNAc(2-4) Man₃GlcNAc₂, including antibodies comprisingNANA (1-4)Gal(1-4)GlcNAc(2-4) Man₃GlcNAc₂,NGNA(1-4)Gal(1-4)GlcNAc(2-4)Man₃GlcNAc₂ or a combination of NANA(1-4)Gal(1-4)GlcNAc(2-4) Man₃GlcNAc₂ and NGNA(1-4)Gal(1-4)GlcNAc(2-4)Man₃GlcNAc₂. In one embodiment, the recombinant glycoprotein willcomprise N-glycans comprising a structure selected from the groupconsisting of SA(1-4)Gal(1-4)GlcNAc(2-4) Man₃GlcNAc₂ and devoid of anyα-2,6 linked SA.

In further embodiments, the host cell that produces glycoproteins thathave predominantly GlcNAcMan₅GlcNAc₂ N-glycans further includes agalactosyltransferase catalytic domain fused to a cellular targetingsignal peptide not normally associated with the catalytic domain andselected to target the galactosyltransferase activity to the ER or Golgiapparatus of the host cell. Passage of the recombinant glycoproteinthrough the ER or Golgi apparatus of the host cell produces arecombinant glycoprotein comprising predominantly theGalGlcNAcMan₅GlcNAc₂ glycoform.

In a further embodiment, the immediately preceding host cell thatproduced glycoproteins that have predominantly the GalGlcNAcMan₅GlcNAc₂N-glycans further includes a sialyltransferase catalytic domain fused toa cellular targeting signal peptide not normally associated with thecatalytic domain and selected to target sialyltransferase activity tothe ER or Golgi apparatus of the host cell. Passage of the recombinantglycoprotein through the ER or Golgi apparatus of the host cell producesa recombinant glycoprotein comprising a SAGalGlcNAcMan₅GlcNAc₂ glycoform(for example NANAGalGlcNAcMan₅GlcNAc₂ or NGNAGalGlcNAcMan₅GlcNAc₂ or amixture thereof).

Any of the preceding host cells can further include one or more sugartransporters such as UDP-GlcNAc transporters (for example, Kluyveromyceslactis and Mus musculus UDP-GlcNAc transporters), UDP-galactosetransporters (for example, Drosophila melanogaster UDP-galactosetransporter), and CMP-sialic acid transporter (for example, human sialicacid transporter). Because lower eukaryote host cells such as yeast andfilamentous fungi lack the above transporters, it is preferable thatlower eukaryote host cells such as yeast and filamentous fungi begenetically engineered to include the above transporters.

Further, any of the preceding host cells can be further manipulated toincrease N-glycan occupancy. See e.g., Gaulitzek et al., Biotechnol.Bioengin. 103:1164-1175 (2009); Jones et al., Biochim. Biospyhs. Acta1726:121-137 (2005); WO2006/107990. In one embodiment, any of thepreceding host cells can be further engineered to comprise at least onenucleic acid molecule encoding a heterologous single-subunitoligosaccharyltransferase (for example, Leishmania sp. STT3A protein,STT3B protein, STT3C protein, STT3D protein or combinations thereof) anda nucleic acid molecule encoding the heterologous glycoprotein, andwherein the host cell expresses the endogenous host cell genes encodingthe proteins comprising the endogenous OTase complex. In one embodiment,any of the preceding host cells can be further engineered to comprise atleast one nucleic acid molecule encoding a Leishmania sp. STT3D proteinand a nucleic acid molecule encoding the heterologous glycoprotein, andwherein the host cell expresses the endogenous host cell genes encodingthe proteins comprising the endogenous OTase complex.

Host cells further include lower eukaryote cells (e.g., yeast such asPichia pastoris) that are genetically engineered to produceglycoproteins that do not have α-mannosidase-resistant N-glycans. Thiscan be achieved by deleting or disrupting one or more of theβ-mannosyltransferase genes (e.g., BMT1, BMT2, BMT3, and BMT4) (See,U.S. Published Patent Application No. 2006/0211085) and glycoproteinshaving phosphomannose residues by deleting or disrupting one or both ofthe phosphomannosyl transferase genes PNO1 and MNN4B (See for example,U.S. Pat. Nos. 7,198,921 and 7,259,007), which in further aspects canalso include deleting or disrupting the MNN4A gene. Disruption includesdisrupting the open reading frame encoding the particular enzymes ordisrupting expression of the open reading frame or abrogatingtranslation of RNAs encoding one or more of the β-mannosyltransferasesand/or phosphomannosyltransferases using interfering RNA, antisense RNA,or the like. Further, cells can produce glycoproteins withα-mannosidase-resistant N-glycans through the addition of chemicalinhibitors or through modification of the cell culture condition. Thesehost cells can be further modified as described above to produceparticular N-glycan structures.

Host cells further include lower eukaryote cells (e.g., yeast such asPichia pastoris) that are genetically modified to controlO-glycosylation of the glycoprotein by deleting or disrupting one ormore of the protein O-mannosyltransferase (Dol-P-Man:Protein (Ser/Thr)Mannosyl Transferase genes) (PMTs) (See U.S. Pat. No. 5,714,377) orgrown in the presence of Pmtp inhibitors and/or an α-mannosidase asdisclosed in Published International Application No. WO 2007/061631, orboth. Disruption includes disrupting the open reading frame encoding thePmtp or disrupting expression of the open reading frame or abrogatingtranslation of RNAs encoding one or more of the Pmtps using interferingRNA, antisense RNA, or the like. The host cells can further include anyone of the aforementioned host cells modified to produce particularN-glycan structures.

Pmtp inhibitors include but are not limited to a benzylidenethiazolidinediones. Examples of benzylidene thiazolidinediones that canbe used are 5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid;5-[[3-(1-Phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticAcid; and5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineaceticacid.

In particular embodiments, the function or expression of at least oneendogenous PMT gene is reduced, disrupted, or deleted. For example, inparticular embodiments the function or expression of at least oneendogenous PMT gene selected from the group consisting of the PMT1,PMT2, PMT3, and PMT4 genes is reduced, disrupted, or deleted; or thehost cells are cultivated in the presence of one or more PMT inhibitors.In further embodiments, the host cells include one or more PMT genedeletions or disruptions and the host cells are cultivated in thepresence of one or more Pmtp inhibitors. In particular aspects of theseembodiments, the host cells also express a secreted α-1,2-mannosidase.

PMT deletions or disruptions and/or Pmtp inhibitors controlO-glycosylation by reducing O-glycosylation occupancy, that is, byreducing the total number of O-glycosylation sites on the glycoproteinthat are glycosylated. The further addition of an α-1,2-mannsodase thatis secreted by the cell controls O-glycosylation by reducing the mannosechain length of the O-glycans that are on the glycoprotein. Thus,combining PMT deletions or disruptions and/or Pmtp inhibitors withexpression of a secreted α-1,2-mannosidase controls O-glycosylation byreducing occupancy and chain length. In particular circumstances, theparticular combination of PMT deletions or disruptions, Pmtp inhibitors,and α-1,2-mannosidase is determined empirically as particularheterologous glycoproteins (Fabs and antibodies, for example) may beexpressed and transported through the Golgi apparatus with differentdegrees of efficiency and thus may require a particular combination ofPMT deletions or disruptions, Pmtp inhibitors, and α-1,2-mannosidase. Inanother aspect, genes encoding one or more endogenousmannosyltransferase enzymes are deleted. This deletion(s) can be incombination with providing the secreted α-1,2-mannosidase and/or PMTinhibitors or can be in lieu of providing the secreted α-1,2-mannosidaseand/or PMT inhibitors.

Thus, the control of O-glycosylation can be useful for producingparticular glycoproteins in the host cells disclosed herein in bettertotal yield or in yield of properly assembled glycoprotein. Thereduction or elimination of O-glycosylation appears to have a beneficialeffect on the assembly and transport of whole antibodies and Fabfragments as they traverse the secretory pathway and are transported tothe cell surface. Thus, in cells in which O-glycosylation is controlled,the yield of properly assembled antibodies or Fab fragments is increasedover the yield obtained in host cells in which O-glycosylation is notcontrolled.

To reduce or eliminate the likelihood of N-glycans and O-glycans withβ-linked mannose residues, which are resistant to α-mannosidases, therecombinant glycoengineered Pichia pastoris host cells are geneticallyengineered to eliminate glycoproteins having α-mannosidase-resistantN-glycans by deleting or disrupting one or more of theβ-mannosyltransferase genes (e.g., BMT1, BMT2, BMT3, and BMT4) (See,U.S. Pat. No. 7,465,577 and U.S. Pat. No. 7,713,719). The deletion ordisruption of BMT2 and one or more of BMT1, BMT3, and BMT4 also reducesor eliminates detectable cross reactivity to antibodies against hostcell protein.

Yield of glycoprotein can in some situations be improved byoverexpressing nucleic acid molecules encoding mammalian or humanchaperone proteins or replacing the genes encoding one or moreendogenous chaperone proteins with nucleic acid molecules encoding oneor more mammalian or human chaperone proteins. In addition, theexpression of mammalian or human chaperone proteins in the host cellalso appears to control O-glycosylation in the cell. Thus, furtherincluded are the host cells herein wherein the function of at least oneendogenous gene encoding a chaperone protein has been reduced oreliminated, and a vector encoding at least one mammalian or humanhomolog of the chaperone protein is expressed in the host cell. Alsoincluded are host cells in which the endogenous host cell chaperones andthe mammalian or human chaperone proteins are expressed. In furtheraspects, the lower eukaryotic host cell is a yeast or filamentous fungihost cell. Examples of the use of chaperones of host cells in whichhuman chaperone proteins are introduced to improve the yield and reduceor control O-glycosylation of recombinant proteins has been disclosed inPublished International Application No. WO 2009105357 and WO2010019487(the disclosures of which are incorporated herein by reference). Likeabove, further included are lower eukaryotic host cells wherein, inaddition to replacing the genes encoding one or more of the endogenouschaperone proteins with nucleic acid molecules encoding one or moremammalian or human chaperone proteins or overexpressing one or moremammalian or human chaperone proteins as described above, the functionor expression of at least one endogenous gene encoding a proteinO-mannosyltransferase (PMT) protein is reduced, disrupted, or deleted.In particular embodiments, the function of at least one endogenous PMTgene selected from the group consisting of the PMT1, PMT2, PMT3, andPMT4 genes is reduced, disrupted, or deleted.

In addition, O-glycosylation may have an effect on an antibody or Fabfragment's affinity and/or avidity for an antigen. This can beparticularly significant when the ultimate host cell for production ofthe antibody or Fab is not the same as the host cell that was used forselecting the antibody. For example, O-glycosylation might interferewith an antibody's or Fab fragment's affinity for an antigen, thus anantibody or Fab fragment that might otherwise have high affinity for anantigen might not be identified because O-glycosylation may interferewith the ability of the antibody or Fab fragment to bind the antigen. Inother cases, an antibody or Fab fragment that has high avidity for anantigen might not be identified because O-glycosylation interferes withthe antibody's or Fab fragment's avidity for the antigen. In thepreceding two cases, an antibody or Fab fragment that might beparticularly effective when produced in a mammalian cell line might notbe identified because the host cells for identifying and selecting theantibody or Fab fragment was of another cell type, for example, a yeastor fungal cell (e.g., a Pichia pastoris host cell). It is well knownthat O-glycosylation in yeast can be significantly different fromO-glycosylation in mammalian cells. This is particularly relevant whencomparing wild type yeast O-glycosylation with mucin-type ordystroglycan type O-glycosylation in mammals. In particular cases,O-glycosylation might enhance the antibody or Fab fragments affinity oravidity for an antigen instead of interfere with antigen binding. Thiseffect is undesirable when the production host cell is to be differentfrom the host cell used to identify and select the antibody or Fabfragment (for example, identification and selection is done in yeast andthe production host is a mammalian cell) because in the production hostthe O-glycosylation will no longer be of the type that caused theenhanced affinity or avidity for the antigen. Therefore, controllingO-glycosylation can enable use of the materials and methods herein toidentify and select antibodies or Fab fragments with specificity for aparticular antigen based upon affinity or avidity of the antibody or Fabfragment for the antigen without identification and selection of theantibody or Fab fragment being influenced by the O-glycosylation systemof the host cell. Thus, controlling O-glycosylation further enhances theusefulness of yeast or fungal host cells to identify and selectantibodies or Fab fragments that will ultimately be produced in amammalian cell line.

Those of ordinary skill in the art would further appreciate andunderstand how to utilize the methods and materials described herein incombination with other Pichia pastoris and yeast cell lines that havebeen genetically engineered to produce specific N-glycans or sialylatedglycoproteins, such as, but, not limited to, the host organisms and celllines described above that have been genetically engineered to producespecific galactosylated or sialylated forms. See, for example, U.S.Publication No. 2006-0286637, Production of Sialylated N-Glycans inLower Eukaryotes, in which the pathway for galactose uptake andutilization as a carbon source has been genetically modified, thedescription of which is incorporated herein as if set forth at length.

Additionally, the methods herein can be used to produce the abovedescribed recombinant Fc-containing polypeptides in other lowereukaryotic cell lines that do not have α-2,3 sialyltransferase activitybut which have been engineered to produce human-like and humanglycoproteins comprising α-2,3-sialyltransferase activity. The methodscan also be used to produce the above described recombinantFc-containing polypeptides in eukaryotic cell lines in which productionof sialylated N-glycans is an innate feature.

Levels of α-2,3 and α-2,6 linked sialic acid on the Fc-containingpolypeptides can be measured using well known techniques includingnuclear magnetic resonance (NMR), normal phase high performance liquidchromatography (HPLC), and high performance anion exchangechromatography with pulsed amperometric detection (HPAEC-PAD).

Production of Fc-Containing Polypeptides

The Fc-containing polypeptides of the invention can be made according toany method known in the art suitable for generating polypeptidescomprising an Fc region having sialylated N-glycans. In one embodiment,the Fc-containing polypeptide is an antibody or an antibody fragment(including, without limitation a polypeptide consisting of or consistingessentially of the Fc region of an antibody). In another embodiment, theFc-containing polypeptide is an immunoadhesin. Methods of preparingantibody, antibody fragments and immunoadhesins are well known in theart. Methods of introducing point mutations into a polypeptide, forexample site directed mutagenesis, are also well known in the art.

In one embodiment, the Fc-containing polypeptides of the invention areexpressed in a host cell that has naturally expresses an α-2,3 sialicacid transferase. In one embodiment, the Fc-containing polypeptides ofthe invention are expressed in a host cell that has been transformedwith a nucleic acid encoding an α-2,3 sialic acid transferase. In oneembodiment the host cell is a mammalian cell. In one embodiment, thehost cell is a lower eukaryotic host cell. In one embodiment, the hostcell is fungal host cell. In one embodiment, the host cell is Pichia sp.In one embodiment, the host cell is Pichia pastoris. In one embodiment,said host cell is capable of producing Fc-polypeptides comprisingsialylated N-glycans, wherein the sialic acid residues in the sialylatedN-glycans contain alpha-2,3 linkages. In one embodiment, said host cellis capable of producing Fc-containing polypeptides, wherein at least30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureselected from the group consisting of SA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂.In one embodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 80% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In any of the above embodiments, the SA couldbe NANA or NGNA, or an analog or derivative of NANA or NGNA. In oneembodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc. In one embodiment, the sialic acid residuesin the sialylatd N-glycans are attached exclusively via α-2,3 linkages.

N-Glycan Analysis of Fc Containing Polypeptides

The N-glycan composition of the antibodies produced herein inglycoengineered Pichia pastoris GFI5.0 and GFI6.0 strains can beanalyzed by matrix-assisted laser desorption ionization/time-of-flight(MALDI-TOF) mass spectrometry after release from the antibody withpeptide-N-glycosidase F. Released carbohydrate composition can bequantitated by HPLC on an Allentech Prevail carbo (Alltech Associates,Deerfield Ill.) column.

Methods of Activating Immune Cells

The invention also comprises a method of activating immune cells orenhancing the effector function of immune cells by contacting an immunecell with an Fc-containing polypeptide comprising α-2,3-linked sialicacid.

The invention also comprises a method of activating immune cells orenhancing the effector function of immune cells by contacting an immunecell with an Fc-containing polypeptide comprising an increased amount ofα-2,3-linked sialic acid compared to the amount of α-2,3-linked in aparent polypeptide. In one embodiment, the Fc-containing polypeptide hasone or more of the following properties when compared to the parentFc-containing polypeptide: (a) increased effector function; (b)increased ability to recruit immune cells (such as T cells, B cells,and/or effector cells/macrophages); and (c) increased inflammatoryproperties. In one embodiment, an Fc-containing polypeptide havingincreased inflammatory properties is an Fc-containing polypeptide whichhas increased/enhanced ability to stimulate the secretion offactors/cytokines which cause inflammation, for example, IL-1, IL-6,RANKL and TNF.

In some embodiments of the invention, the amount of α-2,3-linked sialicacid is increased by expressing the Fc-containing polypeptide in a hostcell that has been transformed with a nucleic acid encoding an α-2,3sialyltransferase. In one embodiment, the host cell is a yeast cell. Insome embodiments, the amount of α-2,3-linked sialic acid is furtherincreased by producing the Fc-containing polypeptide under cell cultureconditions which result in increased sialic acid content. In anotherembodiment, the amount of α-2,3-linked sialic acid is increased byintroducing one or more mutations in the Fc region of the Fc-containingpolypeptide. In one embodiment, the mutations are introduced at one ormore locations selected from the group consisting of: 241, 243, 264,265, 267, 296, 301 and 328, wherein the numbering is according to the EUindex as in Kabat. In one embodiment, the mutations are introduced attwo or more locations selected from the group consisting of: 241, 243,264, 265, 267, 296, 301 and 328. In one embodiment, the mutations areintroduced at positions 243 and 264 of the Fc region. In one embodiment,the mutations at positions 243 and 264 are selected from the groupconsisting of: F243A and V264A; F243Y and V264G; F243T and V264G; F243Land V264A; F243L and V264N; and F243V and V264G. In one embodiment, themutations introduced are F243A and V264A. In another embodiment, themutations introduced are: F243A, V264A, S267E, and L328F.

The above described methods of activating immune cells could be used totreat cancer or infectious diseases (such as chronic viral infenctions)or could be used as an adjuvant to a prophylactic or therapeuticvaccine.

In some embodiments of the above described methods, all of the sialicacid residues in the Fc-containing polypeptide are attached exclusivelyvia an α-2,3 linkage. In other embodiments, most of the sialic acidresidues in the Fc-containing polypeptide are attached via an α-2,3linkage. In other embodiments, some of the sialic acid residues in theFc-containing polypeptide are attached via an α-2,3 linkage while othersare attached via an α-2,6 linkage.

In some embodiments of the above described methods, at least 30%, 40%,50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an oligosaccharide structure selected from thegroup consisting of SA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNA₂.

In some embodiments of the above described methods, at least 30%, 40%,50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an oligosaccharide structure selected from thegroup consisting of SA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂.

In some embodiments of the above described methods, at least 30%, 40%,50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an oligosaccharide structure consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 80% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃ GlcNAc₂.

In some embodiments of the above described methods, at least 30%, 40%,50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an oligosaccharide structure selected from thegroup consisting of NANA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂.

In some embodiments of the above described methods, at least 30%, 40%,50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an oligosaccharide structure consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 80% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂.

In some embodiments, the Fc containing polypeptide comprises the aminoacid sequence of SEQ ID NO:6 or SEQ ID NO:7, plus one or more mutationswhich result in an increased amount of sialic acid. In anotherembodiments, the Fc containing polypeptide comprises the amino acidsequence of SEQ ID NO:6 or SEQ ID NO:7, plus one, two, three or fourmutations which result in an increased amount of sialic acid (forexample, mutations at one or more locations selected from the groupconsisting of: 241, 243, 264, 265, 267, 296, 301 and 328, wherein thenumbering is according to the EU index as in Kabat). In one embodiment,the Fc-containing polypeptide comprises the amino acid sequence of SEQID NO: 8 or 9.

In another embodiment, the amount of α-2,3-linked sialic acid isincreased by expressing the Fc-containing polypeptide in a host cellthat has been transformed with a nucleic acid encoding an α-2,3 sialicacid transferase and by introducing one or more mutations in the Fcregion of the Fc-containing polypeptide. In one embodiment the host cellis a yeast cell. The mutation could be any of the Fc mutations describedabove.

The invention also comprises a method of increasing an immune responseto an antigen, comprising: contacting an immune cell with: (i) anantigen and (ii) an Fc-containing polypeptide comprising α-2,3-linkedsialic acid, such that an immune response to the antigen is increased orenhanced. This method could be conducted in vivo (in a subject) or exvivo. In one embodiment, the invention comprises: (i) obtaining immunecells from a patient, (ii) contacting the immune cells with anFc-containing polypeptide comprising α-2,3 linked sialic acid, and (iii)then administering the immune cells to the patient. In one embodiment,the Fc-containing polypeptide comprises an increased amount ofα-2,3-linked sialic acid compared to the amount of α-2,3-linked in aparent polypeptide.

Methods of Treatment

The Fc-containing polypeptides of the invention could be used in thetreatment of diseases or disorders where destruction or elimination oftissue or foreign microorganisms is desired. For example, theFc-containing polypeptides of the invention could be used to treatneoplastic diseases or infectious (e.g., bacterial, viral, fungal oryeast) diseases. Further, the Fc-containing polypeptides of theinvention could be used as vaccine adjuvants.

The invention comprises a method of enhancing an immune response in asubject in need thereof comprising: administering to the subject atherapeutically effective amount of an Fc-containing polypeptidecomprising α-2,3-linked sialic acid. In one embodiment, the subject asan infectious disease. In another embodiment, the subject has aneoplastic disease.

The invention comprises a method of enhancing an immune response in asubject in need thereof comprising: administering to the subject atherapeutically effective amount of an Fc-containing polypeptidecomprising an increased amount of α-2,3-linked sialic acid compared tothe amount of α-2,3-linked in a parent polypeptide. In one embodiment,the subject as an infectious disease. In another embodiment, the subjecthas a neoplastic disease. In some embodiments, the amount ofα-2,3-linked sialic acid is increased by expressing the Fc-containingpolypeptide in a host cell that has been transformed with a nucleic acidencoding an α-2,3 sialic acid transferase. In one embodiment, the hostcell is a yeast cell. In some embodiments, the amount of α-2,3-linkedsialic acid is further increased by producing the Fc-containingpolypeptide under cell culture conditions which result in increasedsialic acid content. In another embodiment, the amount of α-2,3-linkedsialic acid is increased by introducing one or more mutations in the Fcregion of the Fc-containing polypeptide. In one embodiment, themutations are introduced at one or more locations selected from thegroup consisting of: 241, 243, 264, 265, 267, 296, 301 and 328, whereinthe numbering is according to the EU index as in Kabat. In oneembodiment, the mutations are introduced at two or more locationsselected from the group consisting of: 241, 243, 264, 265, 267, 296, 301and 328. In one embodiment, the mutations are introduced at positions243 and 264 of the Fc region. In one embodiment, the mutations atpositions 243 and 264 are selected from the group consisting of F243Aand V264A; F243Y and V264G; F243T and V264G; F243L and V264A; F243L andV264N; and F243V and V264G. In one embodiment, the mutations introducedare F243A and V264A. In another embodiment, the mutations introducedare: F243A, V264A, S267E, and L328F. In another embodiment, the amountof of α-2,3-linked sialic acid is increased by expressing theFc-containing polypeptide in a host cell that has been transformed witha nucleic acid encoding an α-2,3 sialic acid transferase and byintroducing one or more mutations in the Fc region of the Fc-containingpolypeptide. The mutation could be any of the Fc mutations describedherein.

In some embodiments of the above described methods of treatment, all ofthe sialic acid residues in the Fc-containing polypeptide are attachedexclusively via an α-2,3 linkage. In other embodiments, most of thesialic acid residues in the Fc-containing polypeptide are attached viaan α-2,3 linkage. In other embodiments, some of the sialic acid residuesin the Fc-containing polypeptide are attached via an α-2,3 linkage whileothers are attached via an α-2,6 linkage.

In some embodiments, at least 30%, 40%, 50%, 60%, 70% of the N-glycanson the Fc-containing polypeptide comprise an oligosaccharide structureselected from the group consisting ofSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In some embodiments, at least30%, 40%, 50%, 60%, 70% of the N-glycans on the Fc-containingpolypeptide comprise an oligosaccharide structure consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 80% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In some embodiments, at least 30%, 40%, 50%,60%, 70% of the N-glycans on the Fc-containing polypeptide comprise anoligosaccharide structure consisting of NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Inone embodiment, at least 80% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂.

In one embodiment, the Fc containing polypeptide comprises the aminoacid sequence of SEQ ID NO: 6 or SEQ ID NO:7. In one embodiment, the Fccontaining polypeptide comprises the amino acid sequence of SEQ ID NO:6or SEQ ID NO:7, plus one or more mutations which result in an increasedamount of sialic acid. In one embodiment, the Fc containing polypeptidecomprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7, plusone, two, three or four mutations which result in an increased amount ofsialic acid (for example, mutations at one or more locations selectedfrom the group consisting of: 241, 243, 264, 265, 267, 296, 301 and 328,wherein the numbering is according to the EU index as in Kabat). In someembodiment, the mutations are: F243A/V264A; F243Y/V264G; F243T/V264G;F243L/V264A; F243L/V264N; F243V/V264G; F243A/V264A/S267E/L328F.

In one embodiment, the Fc containing polypeptide comprises the aminoacid sequence of SEQ ID NO:8 or SEQ ID NO:9.

In some embodiments of the above described methods, the Fc-containingpolypeptide has one or more of the following properties when compared toa parent Fc-containing polypeptide: (a) increased effector function; (b)increased ability to recruit immune cells (such as T cells, B cells, andor effector cells/macrophages); and (c) increased inflammatoryproperties.

In one embodiment, the invention comprises a method of enhancing animmune response in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of an Fc-containingpolypeptide comprising sialylated N-glycans, wherein the sialic acidresidues in the sialylated N-glycans contain α-2,3 linkages, and whereinat least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureselected from the group consisting ofSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, the subjecthas, or is at risk of developing, an infectious disease or a neoplasticdisease. In one embodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90%of the N-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, at least 30%, 40%,50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 80% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 30%, 40%, 50%,60%, 70%, 80% or 90% of the N-glycans on the Fc-containing polypeptidecomprise an N-linked oligosaccharide structure consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 80% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, the Fc polypeptidecomprises N-glycans at a position that corresponds to the Asn297 site ofa full-length heavy chain antibody, wherein the numbering is accordingto the EU index as in Kabat. In one embodiment, the N-glycans lackfucose. In another embodiment, the N-glycans further comprise a corefucose. In one embodiment, all of the sialic acid residues in theFc-containing polypeptide are attached exclusively via an α-2,3 linkage.

In one embodiment, the invention comprises a method of enhancing animmune response in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of an Fc-containingpolypeptide comprising sialylated N-glycans, wherein the sialic acidresidues in the sialylated N-glycans contain α-2,3 linkages, and whereinat least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureselected from the group consisting ofSA(1-4)Gal(1-4)GlcNAc(1-4)Man(>=3)GlcNAc2. In one embodiment at least30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an oligosaccharide structure selectedfrom the group consisting of SA(1-3)Gal(1-3)GlcNAc(1-3)Man3GlcNAc2. Inone embodiment, the sialic acid residues in the sialylatd N-glycans areattached exclusively via α-2,3 linkages. In one embodiment, the Fcpolypeptide comprises N-glycans at a position that corresponds to theAsn297 site of a full-length heavy chain antibody, wherein the numberingis according to the EU index as in Kabat. In one embodiment, theN-glycans lack fucose. In another embodiment, the N-glycans furthercomprise a core fucose.

In one embodiment, the invention comprises a method of enhancing animmune response in a subject in need thereof comprising administering tothe subject a therapeutically effective amount of an Fc-containingpolypeptide comprising sialylated N-glycans, wherein the sialic acidresidues in the sialylated N-glycans contain α-2,3 linkages, and whereinat least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureselected from the group consisting ofSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, all of thesialic acid residues in the Fc-containing polypeptide are attachedexclusively via an α-2,3 linkage. In one embodiment, the N-glycans lackfucose. In another embodiment, the N-glycans further comprise a corefucose. In one embodiment, the Fc polypeptide is an antibody or antibodyfragment comprising sialylated N-glycans. In one embodiment, the Fcpolypeptide comprises N-glycans at a position that corresponds to theAsn297 site of a full-length heavy chain antibody, wherein the numberingis according to the EU index as in Kabat. In one embodiment, the Fcpolypeptide is an antibody or antibody fragment comprising or consistingessentially of SEQ ID NO:6 or SEQ ID NO:7. In one embodiment theFc-containing polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 6 or SEQ ID NO: 7, plus one or more mutationswhich result in an increased amount of sialic acid when compared to theamount of sialic acid in a parent polypeptide. In one embodiment theFc-containing polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 6 or SEQ ID NO: 7, plus one, two, three or fourmutations which result in an increased amount of sialic acid whencompared to the amount of sialic acid in a parent polypeptide. In oneembodiment, the parent polypeptide comprises the amino acid sequence ofSEQ ID NO:6 or SEQ ID NO:7. In one embodiment, the Fc-containingpolypeptide is an antibody or antibody fragment comprising mutations atpositions 243 and 264 of the Fc region wherein the numbering isaccording to EU index as in Kabat. In one embodiment, the mutations areF243A and V264A.

In one embodiment, the Fc-containing polypeptide of the invention willbe administered a dose of between 1 to 100 milligrams per kilograms ofbody weight. In one embodiment, the Fc-containing polypeptide of theinvention will be administered a dose of between 0.001 to 10 milligramsper kilograms of body weight. In one embodiment, the Fc-containingpolypeptide of the invention will be administered a dose of between0.001 to 0.1 milligrams per kilograms of body weight. In one embodiment,the Fc-containing polypeptide of the invention will be administered adose of between 0.001 to 0.01 milligrams per kilograms of body weight.

The invention comprises a method of boosting immunogenicity duringvaccination (either prophylactic or therapeutic) comprising:administering to the subject a therapeutically effective amount of anFc-containing polypeptide comprising α-2,3-linked sialic acid. In oneembodiment, the Fc-containing polypeptide is an antibody orimmunoadhesin that recognizes a viral or bacterial antigen. In oneembodiment, the Fc-containing polypeptide comprises an an increasedamount of α-2,3-linked sialic acid compared to the amount ofα-2,3-linked in a parent polypeptide. The amount of sialic acid in anFc-containing polypeptide can be increased using any of the method,including the methods disclosed above.

The invention comprises a method of boosting immunogenicity duringvaccination (either prophylactic or therapeutic) comprising:administering to the subject a therapeutically effective amount of anFc-containing polypeptide comprising sialylated N-glycans, wherein thesialic acid residues in the sialylated N-glycans contain α-2,3 linkages,and wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA(1-4)Gal(1-4)GlcNAc(1-4)Man(>=3)GlcNAc2. In one embodiment at least30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an oligosaccharide structure selectedfrom the group consisting of SA(1-3)Gal(1-3)GlcNAc(1-3)Man3GlcNAc2. Inone embodiment, all of the sialic acid residues in the Fc-containingpolypeptide are attached exclusively via an α-2,3 linkage. In oneembodiment, the Fc polypeptide comprises N-glycans at a position thatcorresponds to the Asn297 site of a full-length heavy chain antibody,wherein the numbering is according to the EU index as in Kabat. In oneembodiment, the N-glycans lack fucose. In another embodiment, theN-glycans further comprise a core fucose. In one embodiment, theFc-containing polypeptide binds a viral or bacterial antigen.

The invention also comprises the use of an Fc-containing polypeptidecomprising α-2,3-linked sialic acid as a vaccine adjuvant. In oneembodiment, at least 30%, 40%, 50%, 60%, 70% of the N-glycans on theFc-containing polypeptide comprise an oligosaccharide structure selectedfrom the group consisting of SA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂.

The invention also comprises the use of an Fc-containing polypeptide avaccine adjuvant. In one embodiment, the Fc-containing polypeptidecomprises sialylated N-glycans, wherein the sialic acid residues in thesialylated N-glycans contain α-2,3 linkages, and wherein at least 30%,40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA(1-4)Gal(1-4)GlcNAc(1-4)Man(>=3)GlcNAc2. Inone embodiment at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an oligosaccharidestructure selected from the group consisting ofSA(1-3)Gal(1-3)GlcNAc(1-3)Man3GlcNAc2. In one embodiment, all of thesialic acid residues in the Fc-containing polypeptide are attachedexclusively via an α-2,3 linkage. In one embodiment, the N-glycans lackfucose. In another embodiment, the N-glycans further comprise a corefucose. In one embodiment, the Fc polypeptide comprises N-glycans at aposition that corresponds to the Asn297 site of a full-length heavychain antibody, wherein the numbering is according to the EU index as inKabat. In one embodiment, the Fc-containing polypeptide binds a viral orbacterial antigen.

In some embodiments, the Fc-containing polypeptide of the invention maybe combined with a second therapeutic agent or treatment modality. Insome embodiments, the Fc-containing polypeptide of the invention(comprising α-2,3-linked sialic acid) may be combined with anothertherapeutic antibody useful for the treatment of cancer or infectiousdisease.

In some embodiments, the Fc-containing polypeptide of the invention(comprising α-2,3-linked sialic acid) is combined with a vaccine toprevent or treat cancer or infectious disease. As a non-limitingexample, the Fc-containing polypeptide of the invention (comprisingα-2,3-linked sialic acid) is combined with a protein, peptide or DNAvaccine containing one or more antigens which are relevant to the canceror infection to be treated, or a vaccine comprising of dendritic cellspulsed with such an antigen. Another embodiment includes the use of theFc-containing polypeptide of the invention (comprising α-2,3-linkedsialic acid) with (attenuated) cancer cell or whole virus vaccines.

Methods of Increasing the Effector Function of an Fc-ContainingPolypeptide

The invention also comprises a method of increasing the effectorfunction or inflammatory properties of an Fc containing polypeptide: (i)selecting a parent Fc-containing polypeptide and (ii) adding orincreasing the amount of, α-2,3-linked sialic acid (for exampleSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂, wherein the sialic acid residuesare exclusively attached to galactose through an α-2,3 linkage) in theparent Fc-containing polypeptide. In one embodiment, the parent Fccontaining polypeptide is a polypeptide that is useful in treating aninfectious disease or a neoplastic disease, or that can be used as avaccine adjuvant.

The invention also comprising a method of increasing the anti-tumorpotency of an Fc-containing polypeptide comprising: (i) selecting aparent Fc-containing polypeptide and (ii) adding or increasing theamount of α-2,3-linked sialic acid (for exampleSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂), wherein the sialic acid residuesare exclusively attached to galactose through an α-2,3 linkage in theparent Fc-containing polypeptide.

The invention also comprising a method of increasing the anti-tumorpotency of an Fc-containing polypeptide comprising: (i) selecting aparent Fc-containing polypeptide and (ii) expressing said Fc-containingpolypeptide in a host cell that has been transformed with a nucleic acidencoding an α-2,3 sialic acid transferase. In one embodiment the hostcell is a mammalian cell. In one embodiment, the host cell is a lowereukaryotic host cell. In one embodiment, the host cell is fungal hostcell. In one embodiment, the host cell is Pichia sp. In one embodiment,the host cell is Pichia pastoris. In one embodiment, said host cell iscapable of producing Fc-polypeptides comprising sialylated N-glycans,wherein the sialic acid residues in the sialylated N-glycans containalpha-2,3 linkages. In one embodiment, said host cell is capable ofproducing Fc-containing polypeptides, wherein at least 30%, 40%, 50%,60%, 70%, 80% or 90% of the N-glycans on the Fc-containing polypeptidecomprise an N-linked oligosaccharide structure selected from the groupconsisting of SA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, atleast 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureconsisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 80%of the N-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In any of the above embodiments, the SA couldbe NANA or NGNA, or an analog or derivative of NANA or NGNA. In oneembodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure consisting of NANA₂Gal₂GlcNAc₂Man₃GlcNAc. Inone embodiment, all of the sialic acid residues in the Fc-containingpolypeptide are attached exclusively via an α-2,3 linkage. In oneembodiment, the N-glycans lack fucose. In another embodiment, theN-glycans further comprise a core fucose.

Biological Targets

It should be noted that while, in the examples that follow, Applicantsexemplifiy the materials and methods of the invention using IgG1antibodies having sequences similar to those for commercially availableanti-TNF antibodies, the invention is not limited to the disclosedantibodies. Those of ordinary skill in the art would recognize andappreciate that the materials and methods herein could be used toproduce any Fc-containing polypeptide, or bioactive form thereof, forwhich the characteristics of enhanced effector function cells would bedesirable. It should further be noted that there is no restriction as tothe type of Fc-containing polypeptide or antibody so produced by theinvention. The Fc region of the Fc-containing polypeptide could be froman IgA, IgD, IgE, IgG or IgM. In one embodiment, the Fc region of theFc-containing polypeptide is from an IgG, including IgG1, IgG2, IgG3 orIgG4. In one embodiment, the Fc region of the Fc-containing polypeptideis from an IgG1. In one embodiment, the Fc region of the Fc-containingpolypeptide is from an IgG1. In specific embodiments, antibodies orantibody fragments produced by the materials and methods herein can behumanized, chimeric or human antibodies.

In some embodiments, the Fc-containing polypeptides of the inventionwill bind to a biological target that is involved in neoplastic disease(i.e., cancer).

In some embodiments, the Fc-containing polypeptide of the invention willbind to an antigen selected from HER2, HERS, EGF, EGFR, VEGF, VEGFR,IGFR, PD-1, PD-1L, BTLA, CTLA-4, GITR, mTOR, CS1, CD20, CD22, CD27,CD28, CD30, CD33, CD40, CD52, CD137, CAl25, MUC1, PEM antigen, Ep-CAM,17-1a, CEA, AFP, HLA-DR, GD2-ganglioside, SK-1 antigen, Lag3, Tim3,CTLA4, TIGIT, SIRPa, ICOS, Trem12, NCR3, HVEM, OX40 and 4-1BB.

In other embodiments, the Fc-containing polypeptide of the inventionwill bind to any pathogenic antigen (for example, a viral or bacterialantigen). In some embodiments, the Fc-containing polypeptide of theinvention will bind to gp120, gp41, Flu HA, an HBV antigen, or an HCVantigen.

Pharmaceutical Formulations

The invention also comprises pharmaceutical formulations comprising anFc-containing polypeptide comprising sialylated N-glycans, wherein thesialic acid residues in the sialylated N-glycans contain α-2,3 linkages,and a pharmaceutically acceptable carrier. In one embodiment, all of thesilaic acid residues in the sialylated N-glycans are attachedexclusively via α-2,3 linkages. In one embodiment, the Fc-containingpolypeptide is an antibody or an antibody fragment or an immunoadhesin.

In one embodiment, the invention relates a pharmaceutical compositioncomprising an Fc-containing polypeptide, wherein at least 30%, 40%, 50%,60%, 70%, 80% or 90% of the N-glycans on the Fc-containing polypeptidecomprise an oligosaccharide structure selected from the group consistingof SA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂, wherein the sialic acidresidues are exclusively attached through an α-2,3 linkage. In oneembodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an oligosaccharidestructure consisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, atleast 80% of the N-glycans on the Fc-containing polypeptide comprise anN-linked oligosaccharide structure selected from the group consisting ofSA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 30%, 40%, 50%,60%, 70%, 80% or 90% of the N-glycans on the Fc-containing polypeptidecomprise an oligosaccharide structure consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least 80% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, the N-glycans lackfucose. In another embodiment, the N-glycans further comprise a corefucose.

In one embodiment, the invention comprises a pharmaceutical formulationcomprising an Fc-containing polypeptide, wherein the Fc-containingpolypeptide comprises sialylated N-glycans, wherein the sialic acidresidues in the sialylated N-glycans contain α-2,3 linkages, and whereinat least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureselected from the group consisting ofSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In one embodiment, at least 30%,40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA₂Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. In oneembodiment, at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure consisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Inone embodiment, at least 80% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, atleast 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureconsisting of NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, at least80% of the N-glycans on the Fc-containing polypeptide comprise anN-linked oligosaccharide structure selected from the group consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In one embodiment, all of the silaic acidresidues in the sialylated N-glycans are attached exclusively via α-2,3linkages. In one embodiment, the N-glycans lack fucose. In anotherembodiment, the N-glycans further comprise a core fucose. In oneembodiment, the N-glycans are attached at a position that corresponds tothe Asn297 site of a full-length heavy chain antibody, wherein thenumbering is according to the EU index as in Kabat.

In one embodiment, the Fc-containing polypeptide has one or more of thefollowing properties when compared to a parent Fc-containingpolypeptide: increased effector function; increased ability to recruitimmune cells; and increased inflammatory properties.

In one embodiment, the Fc-containing polypeptide of the inventioncomprises or consist of the amino acid sequence of SEQ ID NO:6 or SEQ IDNO:7. In another embodiment, the Fc-containing polypeptide of theinvention comprises or consist of the amino acid sequence of SEQ ID NO:6or SEQ ID NO:7, plus one or more mutations which result in an increasedamount of sialic acid when compared to the amount of sialic acid in aparent Fc-containing polypeptide. In another embodiment, theFc-containing polypeptide of the invention comprises or consist of theamino acid sequence of SEQ ID NO:6 or SEQ ID NO:7, plus one, two, threeor four mutations which result in an increased amount of sialic acidwhen compared to the amount of sialic acid in a parent Fc-containingpolypeptide. In one embodiment, the Fc-containing polypeptide of theinvention comprises or consist of the amino acid sequence of SEQ ID NO:8or SEQ ID NO:9.

As utilized herein, the term “pharmaceutically acceptable” means anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s), approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals and, more particularly, in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which the therapeuticis administered and includes, but is not limited to such sterile liquidsas water and oils. The characteristics of the carrier will depend on theroute of administration.

Pharmaceutical formulations of therapeutic and diagnostic agents may beprepared by mixing with acceptable carriers, excipients, or stabilizersin the form of, e.g., lyophilized powders, slurries, aqueous solutionsor suspensions (see, e.g., Hardman et al. (2001) Goodman and Gilman'sThe Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

The mode of administration can vary. Suitable routes of administrationinclude oral, rectal, transmucosal, intestinal, parenteral;intramuscular, subcutaneous, intradermal, intramedullary, intrathecal,direct intraventricular, intravenous, intraperitoneal, intranasal,intraocular, inhalation, insufflation, topical, cutaneous, transdermal,or intra-arterial.

In certain embodiments, the Fc-containing polypeptides of the inventioncan be administered by an invasive route such as by injection (seeabove). In some embodiments of the invention, the Fc-containingpolypeptides of the invention, or pharmaceutical composition thereof, isadministered intravenously, subcutaneously, intramuscularly,intraarterially, intraarticularly (e.g. in arthritis joints),intratumorally, or by inhalation, aerosol delivery. Administration bynon-invasive routes (e.g., orally; for example, in a pill, capsule ortablet) is also within the scope of the present invention.

In certain embodiments, the the Fc-containing polypeptides of theinvention can be administered by an invasive route such as by injection(see above). In some embodiments of the invention, the Fc-containingpolypeptides of the invention, or pharmaceutical composition thereof, isadministered intravenously, subcutaneously, intramuscularly,intraarterially, intraarticularly (e.g. in arthritis joints),intratumorally, or by inhalation, aerosol delivery. Administration bynon-invasive routes (e.g., orally; for example, in a pill, capsule ortablet) is also within the scope of the present invention.

Compositions can be administered with medical devices known in the art.For example, a pharmaceutical composition of the invention can beadministered by injection with a hypodermic needle, including, e.g., aprefilled syringe or autoinjector.

The pharmaceutical compositions of the invention may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

The pharmaceutical compositions of the invention may also beadministered by infusion. Examples of well-known implants and modulesform administering pharmaceutical compositions include: U.S. Pat. No.4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,447,233,which discloses a medication infusion pump for delivering medication ata precise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments. Many other suchimplants, delivery systems, and modules are well known to those skilledin the art.

Alternately, one may administer the antibody in a local rather thansystemic manner, for example, via injection of the antibody directlyinto an arthritic joint, often in a depot or sustained releaseformulation. Furthermore, one may administer the antibody in a targeteddrug delivery system, for example, in a liposome coated with atissue-specific antibody, targeting, for example, arthritic joint orpathogen-induced lesion characterized by immunopathology. The liposomeswill be targeted to and taken up selectively by the afflicted tissue.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic antibody, the level ofsymptoms, the immunogenicity of the therapeutic antibody, and theaccessibility of the target cells in the biological matrix. Preferably,the administration regimen delivers sufficient therapeutic antibody toeffect improvement in the target disease state, while simultaneouslyminimizing undesired side effects. Accordingly, the amount of biologicdelivered depends in part on the particular therapeutic antibody and theseverity of the condition being treated. Guidance in selectingappropriate doses of therapeutic antibodies is available (see, e.g.,Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd,Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokinesand Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993)Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, MarcelDekker, New York, N.Y.; Baert, et al. (2003) New Engl. J. Med.348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973;Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al.(2000) New Engl. J. Med 342:613-619; Ghosh et al. (2003) New Engl. J.Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.Preferably, a biologic that will be used is derived from the samespecies as the animal targeted for treatment, thereby minimizing anyimmune response to the reagent. In the case of human subjects, forexample, chimeric, humanized and fully human Fc-containing polypeptidesare preferred.

Fc-containing polypeptides can be provided by continuous infusion, or bydoses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly,monthly, bimonthly, quarterly, semiannually, annually etc. Doses may beprovided, e.g., intravenously, subcutaneously, topically, orally,nasally, rectally, intramuscular, intracerebrally, intraspinally, or byinhalation. A total weekly dose is generally at least 0.05 μg/kg bodyweight, more generally at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg,100 μg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25mg/kg, 50 mg/kg or more (see, e.g., Yang et al., New Engl. J. Med.349:427-434 (2003); Herold et al., New Engl. J. Med. 346:1692-1698(2002); Liu et al., J. Neurol. Neurosurg. Psych. 67:451-456 (1999);Portielji et al., Cancer Immunol. Immunother. 52:133-144 (2003). Inother embodiments, an Fc-containing polypeptide Of the present inventionis administered subcutaneously or intravenously, on a weekly, biweekly,“every 4 weeks,” monthly, bimonthly, or quarterly basis at 10, 20, 50,80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, the terms “therapeutically effective amount”,“therapeutically effective dose” and “effective amount” refer to anamount of an Fc-containing polypeptide of the invention that, whenadministered alone or in combination with an additional therapeuticagent to a cell, tissue, or subject, is effective to cause a measurableimprovement in one or more symptoms of a disease or condition or theprogression of such disease or condition. A therapeutically effectivedose further refers to that amount of the Fc-containing polypeptidesufficient to result in at least partial amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously. An effective amount of atherapeutic will result in an improvement of a diagnostic measure orparameter by at least 10%; usually by at least 20%; preferably at leastabout 30%; more preferably at least 40%, and most preferably by at least50%. An effective amount can also result in an improvement in asubjective measure in cases where subjective measures are used to assessdisease severity.

Example 1 Construction of Anti-TNFα Fc Muteins

The preparation of an Fc with two mutations (F243A/V264A) in an anti-TNFmonoclonal antibody in Pichia pastoris was carried out using thesequences and protocols listed below. The heavy and light chainsequences of the parent (wildtype) anti-TNFα antibody are set for the inSEQ ID NOs:1 and 2. The sequence of the heavy chain of the double muteinanti-TNFα antibody is set forth in SEQ ID NO:3. The light chain sequenceof the wt and double mutein anti-TNFα antibodies are identical.

The signal sequence of an alpha-mating factor predomain (SEQ ID NOs: 4and 5) was fused in frame to the end of the light or heavy chain by PCRfusion. The sequence was codon optimized and synthesized by Genscript(GenScript USA Inc., 860 Centennial Ave. Piscataway, N.J. 08854, USA).Both heavy chain and light chain were cloned into antibody expressionvector as similar way of constructing anti-HER2 IgG1 and its Fc muteins.

The heavy and light chains with the fused signal sequence of IgG1 andits muteins were cloned under Pichia pastoris AOX1 promoter and in frontof S. cerevisiae Cyc terminator, respectively. The expression cassetteof the completed heavy and light chains was put together into the finalexpression vector. Genomic insertion into Pichia pastoris was achievedby linearization of the vector with Spe1 and targeted integration intothe Trp2 site. Plasmid pGLY6964 encodes wildtype anti-TNFαIgG1 antibody.Plasmid pGLY7715 endoes the anti-TNF alpha IgG1 F243A/V264A doublemutein.Glycoengineered Pichia GFI6.0 YGLY 22834 was the parental hostfor producing Anti-TNFα Fc muteins. Its genotype is listed as follow:ura5Δ::ScSUC2 och1Δ::lacZ bmt2Δ::lacZ/KlMNN2-2 mnn4L1Δ::lacZ/MmSLC35A3pno1Δ mnn4Δ::lacZADE1:lacZ/NA10/MmSLC35A3/FB8his1Δ::lacZ/ScGAL10/XB33/DmUGTarg1Δ::HIS1/KD53/TC54bmt4Δ::lacZ bmt1Δ::lacZ bmt3Δ::lacZTRP2:ARG1/MmCST/HsGNE/HsCSS/HsSPS/MmST6-33ste13Δ::lacZ/TrMDS1dap2Δ::Nat^(R) TRP5:Hyg^(R)MmCST/HsGNE/HsCSS/HsSPS/MmST6-33Vps10-1Δ::AOX1p_LmST73d. Anti-TNF α Fc mutein expressing plasmid wastransformed into YGLY22834 and generated YGLY23423. YGLY23423 was usedas the production strain to make alpha 2,6 sialylated anti-TNF α Fcmutein.

The abbreviations used to describe the genotypes are commonly known andunderstood by those skilled in the art, and include the followingabbreviations:

-   ScSUC2 S. cerevisiae Invertase-   OCH1 Alpha-1,6-mannosyltransferase-   KlMNN2-2 K. lactis UDP-GlcNAc transporter-   BMT1 Beta-mannose-transfer (beta-mannose elimination)-   BMT2 Beta-mannose-transfer (beta-mannose elimination)-   BMT3 Beta-mannose-transfer (beta-mannose elimination)-   BMT4 Beta-mannose-transfer (beta-mannose elimination)-   MNN4L1 MNN4-like 1 (charge elimination)-   MmSLC35A3 Mouse homologue of UDP-GlcNAc transporter-   PNO1 Phosphomannosylation of N-glycans (charge elimination)-   MNN4 Mannosyltransferase (charge elimination)-   ScGAL10 UDP-glucose 4-epimerase-   X833 Truncated HsGalT1 fused to ScKRE2 leader-   DmUGT UDP-Galactose transporter-   KD53 Truncated DmMNSII fused to ScMNN2 leader-   TC54 Truncated RnGNTII fused to ScMNN2 leader-   NA₁₀ Truncated HsGNTI fused to PpSEC12 leader-   FB8 Truncated MmMNS1A fused to ScSEC12 leader-   TrMDS1 Secreted T. reseei MNS1-   ADE1 N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR)    synthetase-   MmCST Mouse CMP-sialic acid transporter-   HsGNE Human UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase-   HsCSS Human CMP-sialic acid synthase-   HsSPS Human N-acetylneuraminate-9-phosphate synthase-   MmST6-33 Truncated Mouse α-2,6-sailyl transferase fused to ScKRE2    leader-   LmSTT3d Catalytic subunit of oligosaccharyltransferase from    Leishmania major

Yeast Transformation and Screening

The glycoengineered GS6.0 strain was grown in YPD rich media (yeastextract 1%, peptone 2% and 2% dextrose), harvested in the logarithmicphase by centrifugation, and washed three times with ice-cold 1 Msorbitol. One to five μg of a Spe1 digested plasmid was mixed withcompetent yeast cells and electroporated using a Bio-Rad Gene PulserXcell™ (Bio-Rad, 2000 Alfred Nobel Drive, Hercules, Calif. 94547) presetPichia pastoris electroporation program. After one hour in recovery richmedia at 24° C., the cells were plated on a minimal dextrose media(1.34% YNB, 0.0004% biotin, 2% dextrose, 1.5% agar) plate containing 300μg/ml Zeocin and incubated at 24° C. until the transformants appeared.

Antibody Purification

Purification of secreted antibody can be performed by one of ordinaryskill in the art using available published methods, for example Li etal., Nat. Biotech. 24(2):210-215 (2006), in which antibodies arecaptured from the fermentation supernatant by Protein A affinitychromatography and further purified using hydrophobic interactionchromatography with a phenyl sepharose fast flow resin.

Generation of α-2,3 Sialyated Anti-TNF Double Mutein Antibody

The reagent identified as “α2,3 SA IgG” corresponds to an anti-TNFantibody having the amino acid sequence of SEQ ID NO:2 and SEQ ID NO:3produced in the GFI 6.0 strain described above, which was in vitrotreated with neuraminidase to eliminate the α2,6 linked sialic acid, andfurther in vitro treated with α-2,3 sialyltransferase. Briefly, thepurified antibody (4-5 mg/me was in the formulation buffer comprising6.16 mg sodium chloride, 0.96 mg monobasic sodium phosphate dehydrate,1.53 mg dibasic sodium phosphate dihydrate, 0.30 mg sodium citrate, 1.30mg citric acid monohydrate, 12 mg mannitol, 1.0 mg polysorbate 80 per 1ml adjusted to pH to 5.2. Neuraminidase (10 mU/ml) was added to antibodymixture and incubated at 37° C. for at least 5 hrs or untildesialylation reached completion. The desialylated material was appliedonto CaptoMMC (GE Healthcare) column purification to removeneuraminidase and reformulated in Sialyltransferase buffer (50 mM HepespH 7.2 150 mM NaCl, 2.5 mM CaCl2, 2.5 mM MgCl2, 2.5 mM MnCl2) at 4mg/ml. Mouse α-2,3 sialyltransferase recombinant enzyme expressed inPichia and purified via his-tag was used for α-2,3 sialic acidextension. The enzyme mixture was formulated in PBS in the presence ofProtease Inhibitor Cocktail (Roche™, cat #11873580001) at 1.2 mg/ml.Prior to the sialylation reaction, pepstatin (50 ug/ml), chymostatin (2mg/ml) and 10 mM CMP-Sialic acid were added to the enzyme mixturefollowed by sterilization through 0.2 μm filter. One ml of enzymemixture was added to 10 ml desialylated material. The reaction wascarried out at 37° C. for 8 hrs. The sialylation yield was confirmed bymass determination by ESI-Q-TOF. The final material was purified usingMabSelect (GE Healthcare) and formulated in the buffer described aboveand sterile-filtered (0.2 μm membrane). The glycosylation of the finalmaterial was analyzed by HPLC based 2-AB labeling method. Approximately89% of the N-glycans on the polypeptide comprised an oligosaccharidestructure selected from the group consisting ofNANA₍₁₋₂₎Gal₍₁₋₂₎GlcNAc(2)Man₃GlcNAc₂.

Example 2 Anti-Tumor Activity of α-2,3 Sialylated Fc-ContainingPolypeptides

In order to determine if α-2,3 linked sialylation of an Fc-containingpolypeptide can enhance the effector function of immune cells, theeffect of α-2,3 linked SA IgG was determined using the 4T1 tumor cellline.

A mouse mammary tumor cell line 4T1 [ATCC CRL-2539] stably transfectedwith firefly luciferase [Luc2] was cultured in RPMI-1640 mediumsupplemented with 10% FBS. Eight-week old female BALB/c mice wereimplanted on the ventral side with 3×105 4T1-Luc2 cells by subcutaneousroute. A week after implantation, the tumors were evaluated by3-dimensional measurements using Biopticon Tumorlmager and randomizedinto treatment groups. Groups of five mice each were treated withindicated doses of antibodies in a weekly treatment regimen for 3consecutive weeks. Tumor volumes were monitored weekly and resultsanalyzed using GraphPad Prism software.

In this model an anti-mouse PD1 antagonistic antibody (generatedin-house) was used as a positive control. An isotype antibody and ananti-CD90 (generated in-house) antibody were used as a negativecontrols.

This experiment shows that anti-PD1 treatment activates CD8 cytotoxicresponses and suppresses tumor growth whereas anti-CD90 deletes all Tlymphocyte subsets and allowes for uncontrolled tumor growth (FIGS.1-2). α-2,3 SA IgG dramatically reduces tumor volume.

Treatment of subcutaneous 4T1-Luc2 mammary tumor bearing mice with α-2,3sialylated IgG resulted in a median tumor growth inhibition (“TGI”) of49% when compared to isotype-treated mice (FIG. 3). Anti-PD1 exhibitedstrong TGI (69%) while anti-CD90 showed poor TGI. The in-vivo tumordoubling time [TDT] for isotype-treated group was 3.9 days compared to4.7 days for the α-2,3 sialylated IgG treated group. By this measure, itwould take an average of about 34 days for the tumor to reach ˜1600cubic mm in a α-2,3 sialylated IgG treated animal compared to only about29 days for an isotype-treated animal.

At the end of the study, mice were treated with 150 mg D-luciferin/Kgbody weight, and the mice were euthanized after 10 minutes. The lungswere harvested and imaged using IVIS Spectrum [Caliper Life Sciences].Relative bioluminescence for lung colonization was evaluated usingLiving Image software. While the tumors in isotype-treated animalsexhibit strong tendency to metastasize to the lung [100% metastasisrate], only 20% of the mice show lung colonization when treated withα-2,3 sialylated IgG suggesting an anti-metastatic effect (FIG. 4).Anti-PD1 treatment also exhibits a strong anti-metastatic effect whereasanti-CD90 treatment mice showed remarkable tumor metastasis in all mice(not shown).

Example 3 Adjuvant and Anti-Tumor Activity of Anti-CD40 AgonisticAntibody Having an Increased Amount of α2,3 Sialic Acid

CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily which is expressed on antigen-presenting cells. CD40 agonistshave been shown to trigger immune responses against various tumors andto inhibit the growth of different neoplastic cells, both in vitro andin vivo. It has been shown that an agonistic mAb to CD40, with enhancedbinding to Fc gamma receptor JIB on antigen-presenting cells, increasesactivation of the antigen-presenting cells and thereby promotes anadaptive immune response (Li and Ravetch, Science 333(6045):1030(2011)). It was proposed that agonistic CD40 antibodies require thecoengagement of the inhibitory FcgRIIB, leading to the maturation of DCspromoting the expansion and activation of cytotoxic CD8+ T cells.

In order to study whether an agonistic anti-CD40 mAb with increased Fcgamma receptor IIB binding could benefit from increased α-2,3 sialicacid content at its Fc region, the antibody is modified by introducingmutations F243A/V264A on its Fc region and by expressing the antibody inthe GFI6.0 strain. This antibody is then studied in the 4T1 metastaticbreast cancer model and/or the murine B-cell lymphoma A20 model forturmor regression and overall long-term animal survival.

The 4T1model is described in Example 2. Briefly a mouse mammary tumorcell line 4T1 [ATCC CRL-2539] stably transfected with firefly luciferase[Luc2] is cultured in RPMI-1640 medium supplemented with 10% FBS.Eight-week old female BALB/c mice are implanted on the ventral side with3×105 4T1-Luc2 cells by subcutaneous route. A week after implantation,the tumors are evaluated by 3-dimensional measurements using BiopticonTumorImager and randomized into treatment groups. Groups of five miceeach are treated with indicated doses of the modified anti-CD40 antibodyin a weekly treatment regimen for 3 consecutive weeks. Tumor volumeswere monitored weekly and results analyzed using GraphPad Prismsoftware.

In another model, animals are challenged with murine B-cell lymphomaturmor cell A20 and then treated with the modified anti-CD40 antibody.A20 cells are maintained in RPMI with 10% FBS, 1% Pen Strep, 1 mM SodiumPyruvate, 10 mM HEPES, and 50 μM 2-Mercaptoethanol. BALB/c mice areinjected intravenously with either 200 μg of mouse control IgG, or themodified anti-CD40 antibody. One hour later, 2×107 A20 cells areinoculated subcutaneously. Tumor growth and long-term survival for A20challenged mice are monitered.

Example 4 Effect of α2,3 Sialylated Fc Fragment in a Collagen-AntibodyInduced Arthritis (AIA) Model

MODEL INDUCTION: AIA (Antibody induced arthritis) is induced with acommercial Arthrogen-CIAe arthritogenic monoclonal antibody (purchasedfrom Chondrex) consisting of a cocktail of 5 monoclonal antibodies,clone A2-10 (IgG2a), F10-21 (IgG2a), D8-6 (IgG2a), D1-2G(IgG2b), andD2-112 (IgG2b), that recognize the conserved epitopes on various speciesof type II collagen.

ANIMALS: 10 week old B10.RIII male mice which are susceptible toarthritis induction without additional of co-stimulatory factors wereused. These animals were purchased from Jackson Laboratory.

CLINICAL SCORING: Paw swelling was measured daily post-induction ofarthritis. Each paw was assessed individually and the paw score wasadded to yield the overall disease score: No swelling=0; Digitswelling=1, Digit and paw selling=2; Digit and paw, with Achilles jointinvolvement=3; minimum per mouse score=0, maximum score=12.

STUDY DESIGN: Arthritis was induced by passive transfer of 3 mg ofanti-CII mAb pathogen cocktail IV on day 0.

Groups of Mice were Treated Subcutaneously with Following Reagents:

Group/Reagent Dose α2,3 SA-Fc 50 mpk Deglycosylated Fc 50 mpk AIAControl 50 mpk Naive 50 mpk Group n = 5 for all groups

The reagent identified as “α2,3 Sialyated Fc” corresponds to an Fcfragment comprising the amino acid sequence of SEQ ID NO:9 (butincluding an additional alanine residue at the 5′ position) produced ina Pichia pastoris strain YGLY31425 having the following geneology:[ura5Δ::ScSUC2 och1Δ::lacZ bmt2Δ::lacZ/KlMNN2-2, mnn4L1Δ::lacZ/MmSLC35A3pno1Δ mnn4Δ::lacZ, ADE1::lacZ/NA10/MmSLC35A3/FB8,his1Δ::lacZ/ScGAL10/XB33/DmUGT, arg1Δ::HIS1/KD53/TC54, bmt4Δ::lacZbmt1Δ::lacZ bmt3Δ::lacZ, TRP2::ARG1/MmCST/HsGNE/HsCSS/HsSPS/rSiaT6-33,TRP5::lacZ/MmCST/HsGNE/HsCSS/HsSPS/rSiaT6-33,ADE8::lacZ-URA5-lacZ/TrMDS1/LmSTT3d, TRP2::Sh ble/hFc double mutein(SEQ2), att1Δ::ScARR3]. The reagent was purified using standard in whichantibodies are captured from the fermentation supernatant by Protein Aaffinity chromatography and further purified using hydrophobicinteraction chromatography with a phenyl sepharose fast flow resin. Theglycosylation of the final material was analyzed by NP-HPLC.Approximately 84% of the N-glycans on the polypeptide comprisedbi-sialylated glycans (NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂) with sialic acidlinked alpha-2,3 to the penultimate galactose residues.

The reagent identified as “Deglycosylated Fc” corresponds to an Fcfragment comprising the amino acid sequence of SEQ ID NO:9 (butincluding an additional alanine residue at the 5′ position) produced inPichia pastoris strain YGLY27893, having the following geneology:[ura5Δ::ScSUC2 och1Δ::lacZ bmt2Δ::lacZ/KlMNN2-2 mnn4L1Δ::lacZ/MmSLC35A3pno1Δ mnn4Δ::lacZADE1:lacZ/NA10/MmSLC35A3/FB8his1Δ::lacZ/ScGAL10/XB33/DmUGTarg1Δ::HIS1/KD53/TC54bmt4Δ::lacZ bmt1Δ::lacZ bmt3Δ::lacZTRP2:ARG1/MmCST/HsGNE/HsCSS/HsSPS/MmST6-33ste13Δ::lacZ/TrMDS1dap2Δ::Nat^(R) TRP5:Hyg^(R)MmCST/HsGNE/HsCSS/HsSPS/MmST6-33 Vps10-1Δ::AOX1p_LmSTT3d TRP2::Sh ble/hFc double mutein (SEQ2)]. The reagent waspurified using standard methods in which antibodies are captured fromthe fermentation supernatant by Protein A affinity chromatography andfurther purified using hydrophobic interaction chromatography with aphenyl sepharose fast flow resin. The protein obtained was treated invitro by PNGase to to remove the N-linked glycan.

The group identified as “AIA control” refers to mice that did notreceive any treatment (other than the administration of the anti-CH mAbpathogen cocktail to induce AIA).

The group identified as “naive” corresponds to mice that did not receivethe anti-CII mAb pathogen cocktail to induce AIA.

All groups of mice were dosed on day 0. The Clinical Score was monitoredfor 10 days.

The results of these experiments are shown in FIG. 5. α2,3 sialylated-Fcdramatically enhanced paw swelling and edema in this inflammation model.

SEQUENCE LISTING SEQ ID NO: Description Sequence 1 heavy chainE  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G  R  S  L  R  L   amino acidS  C  A  A  S  G  F  T  F  D  D  Y  A  M  H  W  V  R  Q  A   sequence ofP  G  K  G  L  E  W  V  S  A  I  T  W  N  S  G  H  I  D  Y   wildtypeA  D  S  V  E  G  R  F  T  I  S  R  D  N  A  K  N  S  L  Y   anti-TNFL  Q  M  N  S  L  R  A  E  D  T  A  V  Y  Y  C  A  K  V  S   alphaY  L  S  T  A  S  S  L  D  Y  W  G  Q  G  T  L  V  T  V  S   antibodyS  A  S  T  K  G  P  S  V  F  P  L  A  P  S  S  K  S  T  S  G  G  T  A  A  L  G  C  L  V  K  D  Y  F  P  E  P  V  T  V  S  W  N  S  G  A  L  T  S  G  V  H  T  F  P  A  V  L  Q  S  S  G  L  Y  S  L  S  S  V  V  T  V  P  S  S  S  L  G  T  Q  T  Y  I  C  N  V  N  H  K  P  S  N  T  K  V  D  K  K  V  E  P  K  S  C  D  K  T  H  T  C  P  P  C  P  A  P  E  L  L  G  G  P  S  V  F  L  F  P  P  K  P  K  D  T  L  M  I  S  R  T  P  E  V  T  C  V  V  V  D  V  S  H  E  D  P  E  V  K  F  N  W  Y  V  D  G  V  E  V  H  N  A  K  T  K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V  L  T  V  L  H  Q  D  W  L  N  G  K  E  Y  K  C  K  V  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  A  K  G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  D  E  L  T  K  N  Q  V  S  L  T  C  L  V  K  G  F  Y  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N  N  Y  K  T  T  P  P  V  L  D  S  D  G  S  F  F  L  Y  S  K  L  T  V  D  K  S  R  W  Q  Q  G  N  V  F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K  S  L  S  L  S  P  G   2 Light chainD  I  Q  M  T  Q  S  P  S  S  L  S  A  S  V  G  D  R  V  T   amino acidI  T  C  R  A  S  Q  G  I  R  N  Y  L  A  W  Y  Q  Q  K  P   sequence ofG  K  A  P  K  L  L  I  Y  A  A  S  T  L  Q  S  G  V  P  S   anti-TNFR  F  S  G  S  G  S  G  T  D  F  T  L  T  I  S  S  L  Q  P   alphaE  D  V  A  T  Y  Y  C  Q  R  Y  N  R  A  P  Y  T  F  G  Q   antibodyG  T  K  V  E  I  K  R  T  V  A  A  P  S  V  F  I  F  P  P  S  D  E  Q  L  K  S  G  T  A  S  V  V  C  L  L  N  N  F  Y  P  R  E  A  K  V  Q  W  K  V  D  N  A  L  Q  S  G  N  S  Q  E  S  V  T  E  Q  D  S  K  D  S  T  Y  S  L  S  S  T  L  T  L  S  K  A  D  Y  E  K  H  K  V  Y  A  C  E  V  T  H  Q  G  L  S  S  P  V  T  K  S  F  N  R  G  E  C   3 Heavy chainE  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G  R  S   amino acidL  R  L  S  C  A  A  S  G  F  T  F  D  D  Y  A  M   sequence ofH  W  V  R  Q  A  P  G  K  G  L  E  W  V  S  A  I   doubleT  W  N  S  G  H  I  D  Y  A  D  S  V  E  G  R  F   mutein anti-T  I  S  R  D  N  A  K  N  S  L  Y  L  Q  M  N  S   TNF alphaL  R  A  E  D  T  A  V  Y  Y  C  A  K  V  S  Y  L   antibodyS  T  A  S  S  L  D  Y  W  G  Q  G  T  L  V  T  V  S  S  A  S  T  K  G  P  S  V  F  P  L  A  P  S  S  K  S  T  S  G  G  T  A  A  L  G  C  L  V  K  D  Y  F  P  E  P  V  T  V  S  W  N  S  G  A  L  T  S  G  V  H  T  F  P  A  V  L  Q  S  S  G  L  Y  S  L  S  S  V  V  T  V  P  S  S  S  L  G  T  Q  T  Y  I  C  N  V  N  H  K  P  S  N  T  K  V  D  K  K  V  E  P  K  S  C  D  K  T  H  T  C  P  P  C  P  A  P  E  L  L  G  G  P  S  V  F  L  A  P  P  K  P  K  D  T  L  M  I  S  R  T  P  E  V  T  C  V  V  A  D  V  S  H  E  D  P  E  V  K  F  N  W  Y  V  D  G  V  E  V  H  N  A  K  T  K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V  L  T  V  L  H  Q  D  W  L  N  G  K  E  Y  K  C  K  V  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  A  K  G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  D  E  L  T  K  N  Q  V  S  L  T  C  L  V  K  G  F  Y  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N  N  Y  K  T  T  P  P  V  L  D  S  D  G  S  F  F  L  Y  S  K  L  T  V  D  K  S  R  W  Q  Q  G  N  V  F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K  S  L  S  L  S  P  G   4 Alpha-matingGAATTCGAAACGATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCT   factor DNACCGCATTAGCT   sequence 5 Alpha-mating MRFPSIFTAVLFAASSALA   factor aminoacid sequence 6 Fc regionT  C  P  P  C  P  A  P  E  L  L  G  G  P  S  V  F  L  F  P   (wt)P  K  P  K  D  T  L  M  I  S  R  T  P  E  V  T  C  V  V  V  D  V  S  H  E  D  P  E  V  K  F  N  W  Y  V  D  G  V  E  V  H  N  A  K  T  K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V  L  T  V  L  H  Q  D  W  L  N  G  K  E  Y  K  C  K  V  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  A  K  G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  D  E  L  T  K  N  Q  V  S  L  T  C  L  V  K  G  F  Y  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N  N  Y  K  T  T  P  P  V  L  D  S  D  G  S  F  F  L  Y  S  K  L  T  V  D  K  S  R  W  Q  Q  G  N  V  F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K  S  L  S  L  S   P  G   7Fc region E  P  K  S  C  D  K  T  H  T  C  P  P  C  P  A  P  E  L  L  (wt) G  G  P  S  V  F  L  F  P  P  K  P  K  D  T  L  M  I  S  R  T  P  E  V  T  C  V  V  V  D  V  S  H  E  D  P  E  V  K  F  N  W  Y  V  D  G  V  E  V  H  N  A  K  T  K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V  L  T  V  L  H  Q  D  W  L  N  G  K  E  Y  K  C  K  V  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  A  K  G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  D  E  L  T  K  N  Q  V  S  L  T  C  L  V  K  G  F  Y  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N  N  Y  K  T  T  P  P  V  L  D  S  D  G  S  F  F  L  Y  S  K  L  T  V  D  K  S  R  W  Q  Q  G  N  V  F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K  S  L  S  L  S  P  G   8 Fc regionT  C  P  P  C  P  A  P  E  L  L  G  G  P  S  V  F  L  A  P   (DM)P  K  P  K  D  T  L  M  I  S  R  T  P  E  V  T  C  V  V  A  D  V  S  H  E  D  P  E  V  K  F  N  W  Y  V  D  G  V  E  V  H  N  A  K  T  K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V  L  T  V  L  H  Q  D  W  L  N  G  K  E  Y  K  C  K  V  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  A  K  G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  D  E  L  T  K  N  Q  V  S  L  T  C  L  V  K  G  F  Y  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N  N  Y  K  T  T  P  P  V  L  D  S  D  G  S  F  F  L  Y  S  K  L  T  V  D  K  S  R  W  Q  Q  G  N  V  F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K  S  L  S  L  S   P  G   9Fc region E  P  K  S  C  D  K  T  H  T  C  P  P  C  P  A  P  E  L  L  (DM) G  G  P  S  V  F  L  A  P  P  K  P  K  D  T  L  M  I  S  R  T  P  E  V  T  C  V  V  A  D  V  S  H  E  D  P  E  V  K  F  N  W  Y  V  D  G  V  E  V  H  N  A  K  T  K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V  L  T  V  L  H  Q  D  W  L  N  G  K  E  Y  K  C  K  V  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  A  K  G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  D  E  L  T  K  N  Q  V  S  L  T  C  L  V  K  G  F  Y  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N  N  Y  K  T  T  P  P  V  L  D  S  D  G  S  F  F  L  Y  S  K  L  T  V  D  K  S  R  W  Q  Q  G  N  V  F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K  S  L  S  L  S  P  G  

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof.

What is claimed: 1) A method of enhancing an immune response in asubject in need thereof comprising administering to the subject atherapeutically effective amount of an Fc-containing polypeptidecomprising sialylated N-glycans, wherein the sialic acid residues in thesialylated N-glycans contain α-2,3 linkages, and wherein at least 30%,40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on the Fc-containingpolypeptide comprise an N-linked oligosaccharide structure selected fromthe group consisting of SA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃ GlcNAc₂. 2) Themethod of claim 1, wherein the subject has, or is at risk of developing,an infectious disease or a neoplastic disease. 3) The method of claim 1or 2, wherein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of theN-glycans on the Fc-containing polypeptide comprise an N-linkedoligosaccharide structure selected from the group consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. 4) The method of any one claims 1-3,wherein the Fc polypeptide is an antibody or antibody fragment. 5) Themethod of any one claim 1-4, wherein the Fc polypeptide is an antibodyfragment consisting essentially of SEQ ID NO:6 or SEQ ID NO:7 6) Themethod of any one of claims 1-4, wherein the Fc-containing polypeptideis an antibody or antibody fragment comprising or consisting essentiallyof the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, plus one ormore mutations which result in an increased amount of sialic acid whencompared to the amount of sialic acid in the parent polypeptide. 7) Themethod of claim 6, wherein the Fc-containing polypeptide is an antibodyor antibody fragment comprising mutations at positions 243 and 264 ofthe Fc region wherein the numbering is according to EU index as inKabat. 8) The method of any one of claims 1-7, wherein saidFc-containing polypeptide has one or more of the following propertieswhen compared to a parent Fc-containing polypeptide: a) increasedeffector function b) increased ability to recruit immune cells, and c)increased inflammatory properties. 9) A pharmaceutical formulationcomprising an Fc-containing polypeptide, wherein the Fc-containingpolypeptide comprises sialylated N-glycans, wherein the sialic acidresidues in the sialylated N-glycans contain α-2,3 linkages, and whereinat least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the N-glycans on theFc-containing polypeptide comprise an N-linked oligosaccharide structureselected from the group consisting ofSA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. 10) The pharmaceuticalformulation of claim 9, wherein at least 30%, 40%, 50%, 60%, 70%, 80% or90% of the N-glycans on the Fc-containing polypeptide comprise anN-linked oligosaccharide structure selected from the group consisting ofNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. 11) The pharmaceutical formulation of anyone of claims 9-10, wherein the Fc-containing polypeptide has one ormore of the following properties when compared to a parent Fc-containingpolypeptide: (a) increased effector function; (b) increased ability torecruit immune cells; and (c) increased inflammatory properties. 12) Thepharmaceutical formulation of any one of claims 9-11, wherein theFc-containing polypeptide is an antibody fragment consisting essentiallyof SEQ ID NO:6 or SEQ ID NO:7. 13) The pharmaceutical formulation of anyone of claims 9-11, wherein the Fc-containing polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7, plusone or more mutations which result in an increased amount of sialic acidwhen compared to the amount of sialic acid in a parent polypeptide. 14)The pharmaceutical formulation of claim 13, wherein the Fc-containingpolypeptide is an antibody or antibody fragment comprising mutations atpositions 243 and 264 of the Fc region wherein the numbering isaccording to EU index as in Kabat.